Curcumol derivatives, the compositions containing the said derivatives, and the use of the same in the manufacture of medicaments

ABSTRACT

The present invention provides curcumol derivatives of the following formula (I) or pharmaceutical acceptable salts thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein, Y is selected from the group consisting of ═CHR 2 , —CH 2 R 2 , ═O, 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
              —OH or —OR 1 ;R 1  is selected from H, R, RCO or HO 3 S; and R is selected from the group consisting of H; saturated or unsaturated linear C 1-10  hydrocarbon group and the like; R 2  is selected from the group consisting of F; Cl; Br; I; H; —OH; —OR; —HSO 3  and the like; with the proviso that both R 1  and R 2  are not H. The present invention also provides anti-tumor or antiviral pharmaceutical compositions comprising said derivatives or pharmaceutical acceptable salts thereof. The present invention further provides the use of said derivatives or pharmaceutical acceptable salts thereof in the preparation of a medicament for prophylaxis and/or treatment tumor or an antiviral medicament.

TECHNICAL FIELD

The present invention relates to curcumol derivatives, compositionscontaining the said derivatives, and use of the same in the manufactureof anti-tumor and antiviral medicaments.

TECHNICAL BACKGROUND

Zedoaria is a Zingliberaceae plant Curcuma zedoaria. The rhizome ofCurcuma kwangsiensis S. Lee et C. F. Liang or curcuma aromatica Salisb(RADIX CURCUMAE) is bitter and acrid in tastep, warm-natured and is abletoregulate Qi and promote blodd circulation, remove food retention andalleviate pain. It mainly cures abdominal mass, congestion andamenorrhea, food retention and gas pains. Its valotile oil can be usedto treat uterine cervix cancer. It has been included in the Chinesepharmacopeia of 1977 (Page 463). The active component is the volatileoil with the content being 1%-2.5%. The main components in the oil are avariety of sesquiterpenes: curzerenone, curdione, neocurdione,epicurcumol, curzerene, curcumol, isocurcumol, procurcumenol,dehydrocurdione and the like, totally about more than twenty chemicalcomponents. Curcumol and curdione are the mainly effective components ofcurcumol oil for treating cancer.

Curcumol, also known as curcumenol and turmerol, molecular formula:C₁₅H₂₄O₂, molecular weight: 236.34, [CAS] 4871-97-0, melting point:141-142° C., has the following structure formula:

Subcutaneously injecting of 75 mg/Kg curcumol exhibited a highinhibition rate against mouse sarcoma 37, uterine cervix cancer, andEhrlich's ascites carcinoma (EAC). In patients whose tumor wasremarkably diminished, it could be observed that fibrocytes around thetumor tissue significantly increased. They have a inner layer oflymphocyte. Immune response, for example, phagocytes surrounding thetumor cells, was also observed.

Curcumol has not only a significant anti-tumor effect, but also theeffects of promoting immune response, increasing leucocytes, protectingthe liver, preventing kidney failure, anti-thromb, antibiosis, and thelike. At the same time, it has no observable toxicity, and its sideeffect is relatively low.

Therefore, curcumol is a very useful natural drugs. However, it existsthe following deficiencies:

-   -   1. Poor water solubility, difficult to produce stable medicament        liquid with an appropriate concentration.    -   2. Severe pain when being topically injected or injected into        the neoplasma. Chest distress, flush and dyspneic respiration        and other symptoms will occur when it is injected too fast.    -   3. The variety of tumors to be treated is limited.    -   4. Strong toxicity.

Although the total effective rate reaches more than 70% with respect toearlier period uterine cervix cancer, there still exists the possibilityfor further improving the drug effect.

CONTENTS OF THE INVENTION

An object of the invention is to provide curcumol derivatives havinggood stability and solubility, better partition coefficient between lipophase and aqueous phase, anti-tumor activity and broader spectrum foranti-tumor, better drug effect and less toxicity.

Another object of the invention is to provide a pharmaceuticalcomposition comprising the curcumol derivatives.

A further object of the invention is to provide the use of the curcumolderivatives in the manufacture of medicaments.

The present invention relates to a curcumol derivative with formula I,or a pharmaceutically acceptable salt thereof

wherein, Y is selected from the group consisting of ═CHR², —CH₂R², ═O,

—OH and —OR¹;

R¹ is selected from the group consisting of H, R, RCO and HO₃S;

R is selected from the group consisting of H; saturated or unsaturatedlinear C₁₋₁₀ hydrocarbon group; saturated or unsaturated branched C₃₋₁₀hydrocarbon group; C₃₋₁₀ hydrocarbon ether; C₃₋₁₀ hydrocarbon sulfide;saturated or unsaturated, C₃₋₈ cyclic hydrocarbon group opticallysubstituted by one or more substitutents selected from the groupconsisting of nitro, sulfonic group, halogen atom and hydroxyl group; orC₆₋₁₂ aryl group;

Y¹NY², Y¹CONY², wherein Y¹ is H or C₁₋₈ and Y² is C_(1-8;)

being selected from the following heterocycles:

wherein, P is S, O or N, and Z is one or more substitutents selectedfrom the group consisting of H, hydroxy group, saturated or unsaturatedlinear C₁₋₆ hydrocarbon group, saturated and unsaturated branched C₃₋₆hydrocarbon group, and n=1-3; or

R¹ is the acyl of coffeic acid, gambogic acid, of isogambogic acid, ofneogambogic acid or of glycyrrhizic acid;

R² is selected from the group consisting of F; Cl; Br; I; H; —OH; —OR;—HSO₃; —NO₃; RNH— and R′NR″, wherein, R′ and R″ may be the same ordifferent, and each is selected from the group consisting of the groupsdefined in R and H₂NRNH—;

or R² is selected from the group consisting of pyridyl, pyrrolyl,imidazolyl, triazolyl, tetrazolyl, dioxazolyl, dioxadiazolyl, piperidyl,

With the proviso that both R¹ and R² are not H,

Y . . . is a single bond, when Y is —OH or —OR¹,

When Y is —CHR², the derivative has the general formula II:

wherein, R¹ and R² are defined as above,

R³ is selected from the group consisting of F; Cl; Br; I; H; —OH; —HSO₃;—NO₃; —RNH and R′NR″, wherein, R′ and R″ may be the same or different,and each is selected from the group consisting of the one for definingR; or R′ and R″ may independently be H₂NRNH—;

or R² is selected from the group consisting of pyridyl, pyrrolyl,imidazolyl, triazolyl, tetrazolyl, dioxazolyl, dioxadiazolyl, piperidyl,

In a preferred embodiment, the aryl group is preferably selected fromthe group consisting of Ar—, ArCH₂—, ArCH₂CH₂—, and CH₃ArCH₂CH₂—, Ar— isphenyl, or phenyl group substituted with F, Cl, Br, I, nitro, sulfonicacid group or 1-3 hydroxy groups. When Y is ═CHR², R² is preferably H;and R¹ is preferably HO₃S, propionyl, butyryl, isobutyryl or benzoyl.

In another preferred embodiment, when Y is ═CHR², R² is H, and R¹ isHO₃S or NaO₃S.

The most preferable curcumol derivatives or pharmaceutically acceptablesalts are selected from the group consisting of the following compounds:

III

No. Name of the compounds R¹ R² 1 Curcumol acetate CH₃CO— H 2 Curcumolbutyrate CH₃CH₂CH₂CH₂C— H 3 Curcumol isobutyrate (CH₃)₂CH₂CO— H 4Curcumol benzoate

H 5 Curcumol p-hydroxy aniline H

6 Curcumol p-hydroxy aniline hydrochloride H

7 Curcumol piperazine H

8 Curcumol piperazine hydrochloride H

9 Curcumol heterocyclyl ethylamine H

10 Curcumol heterocyclyl ethylamine hydrochloride H

11 3,4-dihydroxy aniline H

12 3,4-dihydroxy aniline hydrochloride H

13 Curcumol n-butyl amine H CH₃CH₂CH₂CH₂NH— 14 Curcumol n-butyl amine HCH₃CH₂CH₂CH₂NH— hydrochloride 15 Curcumol t-butyl amine H (CH₃)₃CNH— 16Curcumol t-butyl amine H (CH₃)₃CNH— hydrochloride 17 Curcumolmonobromide H —Br 18 Curcumol monohydroxy H —OH compound 19 Curcumolmononitrate H —NO₃ 20 Curcumol sulfonate HSO₃— H 21 Curcumol sodiumNaSO₃— H sulfonate 22 Curcumol acrylate CH₂═CHCO— H 23 Curcumoldiethanolamine H (CH₃CH₂)₂N— 24 Curcumol diethanolamine H (CH₃CH₂)₂N—hydrochloride 25 Curcumol methyl ether CH₃ H 26 Curcumol methyl etherCH₃ —Br bromide 27 Curcumol methyl ether CH₃ CH₃CH₂CH₂CH₂NH— n-butylamine 28 Curcumol methyl ether CH₃ CH₃CH₂CH₂CH₂NH— n-butyl aminehydrochloride 29 Curcumol ethyl ether CH₃CH₂— —NO₃ nitrate

Alternatively, the curcumol derivatives or pharmaceutically acceptablesalts thereof have the following structures:

II

wherein, the groups in the formula are defined as follows: No. Name ofthe compounds R¹ R² R³ 30 Curcumol dibromide H —Br —Br 31 Curcumoldinitrate H —NO₃ —NO₃ 32 Curcumol dihydroxy compound H —OH —OH 33hydrogenated curcumol derivative H H H 34 Curcumol monobromide withoutdouble H H —Br bond 35 Curcumol monohydroxy compound without H H —OHdouble bond 36 Curcumol mononitrate without double bond H H —NO₃ 37Curcumol p-hydroxy aniline without double bond H H

38 Curcumol p-hydroxy aniline hydrochloride without double bond H H

39 Curcumol bis (p-hydroxy aniline) H

40 Curcumol bis (p-hydroxy aniline) hydrochloride H

41 Curcumol bispiperazine H

42 Curcumol bispiperazine hydrochloride H

43 Curcumol piperazine without double bond H H

44 Curcumol piperazine hydrochloride without double bond H H

45 Curcumol methyl ether n-butyl amine CH₃ H CH₃CH₂CH₂CH₂NH withoutdouble bond 46 Curcumol methyl ether n-butyl amine CH₃ H CH₃CH₂CH₂CH₂NHhydrochloride without double bond

In another variant, the curcumol derivatives or the pharmaceuticallyacceptable thereof have the following structures:

Wherein, the groups in the formula are defined as follows:

No. Name of the compounds R¹ Y 47 Curcumol epoxy compound H

48 Curzerenone H O 49 Curzerenone acetate CH₃CO— O

In another most preferred embodiment of the present invention, the mostpreferred curcumol derivatives or pharmaceutical salts thereof areselected from the following compounds:

curcumol p-hydroxy aniline H

curcumol p-hydroxy aniline hydrochloride H

Curcumol propionate CH₃CH₂CO— H Curcumol butyrate CH₃CH₂CH₂C0— HCurcumol isobutyrate (CH₃)₂CH₂CO— H Curcumol benzoate

H Curcumol monobromide H —Br Curcumol sulfonate HSO₃— H Curcumol sodiumsulfonate NaSO₃— H Curcumol pipearzine H

Curcumol pipearzine hydrochloride H

Curcumol n-butyl amine H CH₃CH₂CH₂CH₂NH— Curcumol n-butyl aminehydrochloride H CH₃CH₂CH₂CH₂NH— Curcumol t-butyl amine H (CH₃)₃CNH—Curcumol t-butyl amine hydrochloride H (CH₃)₃CNH—

The present invention also provides an anti-tumor or antiviralpharmaceutical composition comprising a pharmaceutically effectiveamount of the afore-mentioned curcumol derivatives or the pharmaceuticalacceptable salts thereof, and a pharmaceutically acceptable excipientand/or an additive.

The present invention further provides a use of the afore-mentionedcurcumol derivatives or the pharmaceutical acceptable salts thereof inthe manufacture of medicaments for treating and/or preventing tumor.

The present invention also provides a use of the afore-mentionedcurcumol derivatives or pharmaceutical acceptable salts thereof in themanufacture of anti-viral medicaments. The virus is selected from thegroup consisting of HIV virus, influenza virus, hepatitis virus andherpes virus.

Curcumol is a component of oleum curcumae wenchowensis, whose content isrelatively high. After purifying the commercially available oleumcurcumae wenchowensis, curcumol is obtained with relatively high purity(the purity can be higher than 75%, and if necessary, the purity canreach more than 99%). The curcumol of relatively high purity is used asa starting material to be structurally modified. The general methods forthe preparation of curcumol derivatives are introduced as follows:

Preparing Hydrogenated Curcumol Derivative

Curcumol was dissolved in an appropriate organic solvent (such asmethanol and the like). A small amount of hydrogenation catalyst (e.g.,palladium on carbon, and the like) was added. Hydrogen gas wasintroduced at room temperature, under normal pressure with vigorousstirring to provide a curcumol derivative in which the double bondbetween 8- and 12-position is hydrogenated.

Preparing the Ester of Curcumol (the Hydroxy Group at ⁶C)

Due to the presence of a hydroxy group at ⁶C of curcumol, curcumol hasthe general propertiess possessed by alcohols. The esters of curcumolthus can be prepared by general methods: for example, curcumol as astarting material was dissolved in an appropriate organic solvent (forexample, isopropyl ether, DMF, dioxane, toluene, dichlormethane,chloroform, acetic acid, and the like), and reacted with carboxylic acidin the presence of a catalyst (e.g., p-toluenesulfonic acid, the complexof dimethylamine sulfonyl chloride with dimethylamine and DMAP, thecomplex of diphenylamine with DPAT, the complex of DMAP with DPC,organo-titanium, p-toluenesulfonic acid, and the like); or reacted withacyl chloride in the presence of acid binding agents (for example,pyridine, triethylamine, and the like); or reacted with acid anhydrideunder reflux, to provide the corresponding ester of curcumol. Then afterseparation and purification treatment, the ester of curcumol inrelatively high purity was obtained. Unless indicated otherwise, in thefollowing schemes, “r.t.” represents “room temperature”, “hr” represents“hours”.

1. The Esterification of Curcumol with Acyl Chloride

Curcumol was dissolved in chloroform and reacted with equal amount ofmole or an excess amount of acyl chloride in the presence of acidbinding agents (e.g., excessive amount of triethylamine) for severalhours at room temperature or under reflux, the corresponding ester ofcurcumol (R in the following formula is defined as above) was obtained:

2. The Esterification of Curcumol with Acid Anhydride

Curcumol was dissolved in carboxylic acid (generally acetic acid) andrefluxed with an excess amount of acid anhydride for several hours. Theester of curcumol corresponding to the acid anhydride was obtained (R inthe following formula is defined as above):

3. The Esterification of Curcumol with Organic Acid in the Presence ofOrgano-Titanium

Catalyst

Curcumol was dissolved in dichlormethane, and carboxylic acid andcurcumol in substantially equal moles were reacted at room temperaturein the presence of organo-titanium reagent as a catalyst andtrimethylchlorosilane as a cocatalyst, and in the presence of equal moleof acid anhydride as a dehydrating agent, to provide the correspondingester of curcumol with high yields (in the following formula, R¹ is R,and R is defined as above, R²OH is curcumol):

4. The Esterification of Curcumol with Organic Acid Under the Catalysisof DMAP and DPC

Curcumol was dissolved in dioxane and reacted with a slight excess ofcarboxylic acid at 70° C. for 100 hours in the presence of the catalystsDMAP and DPC, to afford the corresponding the ester of curcumol withhigh yield (R in the following formula is defined as above):

5. The Esterification of Curcumol with Organic Acid Catalyzed byp-toluenesulfonic Acid

Curcumol was dissolved in toluene, and reacted with an excess ofcarboxylic acid under reflux in the presence of the catalystp-toluenesulfonic acid while the resulting water was separated, toafford the corresponding ester of curcumol (R in the following formulais defined as above):

6. Curcumol was dissolved in an appropriate organic solvents (e.g., DMF,ethyl acetate or pyridine, etc.), and reacted with thionyl dichloride.After reacting, the mixture was treated with an alkaline solution toafford curcumol sulfate:

Similarly, the above prepared hydrogenated curcumol derivative was usedas a starting material and proceeded with the above reaction schemes 1-6to afford the corresponding esters of hydrogenated curcumol derivatives.

Preparing Curcumol Ether (the Hydroxy Group at ⁶C)

Due to the presence of a hydroxy group at ⁶C of curcumol, curcumol hasthe general properties of alcohols. Curcumol was dissolved in anappropriate organic solvent (e.g., isopropyl ether, DMF, dioxane,toluene, dichlormethane, chloroform), then was reacted with sulfate(ROSO₂OR) or an alkane iodide (RI) in a basic solution to afford thecorresponding ethers. After the separation and the purification, thecurcumol ether in relatively high purity was obtained.

1. Curcumol was dissolved in isopropyl ether and dichlormethane (R inthe following formula was defined as above):

2. Curcumol was dissolved in isopropyl ether and dichlormethane, thenwas reacted with alkane iodide (R in the following formula was definedas above):

Similarly, the above prepared hydrogenated curcumol derivative was usedas a starting material and proceeded with the above reaction schemes 1-2to afford the corresponding ether of hydrogenated curcumol derivative.

Due to the presence of a double bond between ⁸C and ¹²C of curcumol,curcumol has the properties, such as being added or being oxidized, thesame as those of the other alkene compounds. It may be easily subjectedto an addition reaction with halogen, or hydrogen halide to affordcurcumol dihalide, or monohalide; or may be reduced by H₂, LiAlH₄ andthe like to afford the hydrogenated curcumol derivatives, or may beadded by alkyl epoxy compounds to afford curcumol epoxy compounds; ormay be oxidized by oxidizing agents such as potassium permanganate toafford curcumol oxide, dihydroxy compounds.

The halogen atom of the curcumol halide obtained from the above reactioncan be replaced by RNH, —R′NR″(R′, R″, the same or different, are R),H₂NRNH—, wherein R is defined as above, or a heterocyclic group, such aspyridine, pyrole, imidazole, triazole, tetrazole, dioxazole,dioxdiazole, piperidine (as long as there is a nitrogen-hydrogen bondH—N< in the molecule:) or the simple derivatives thereof,

(ortho, meta or para position) (wherein R is defined as above or is H)at room temperature in an appropriate organic solvent to afford thecorresponding amine derivatives.

1. The Preparation of the Adduct of Curcumol with Halogen

Curcumol was dissolved in an appropriate organic solvent (such aschloroform, methanol and the like), and was subjected to an additionreaction with halogen (X₂, X₂ is Cl₂, Br₂, I₂, preferably Br₂) at roomtemperature or below 0° C., to yield a curcumol derivative in which eachof ⁸C and ¹²C were added a halogen atom.

The prepared dihalide tended to eliminate a hydrogen chloride inanhydrous organic solvent under alkaline condition to afford amonohalide in which the double bond between ⁸C and ¹²C was rebuilt.

The above prepared curcumol (the hydroxy group at ⁶C) ester, or curcumol(the hydroxy group at ⁶C) ether (R is defined as above, but R does motinclude the alkene group containing a double bond), as a startingmaterial, was dissolved in an appropriate organic solvent (such aschloroform, methanol and the like), and was subjected to an additionreaction with halogen at room temperature or below 0° C., to afford thecorresponding curcumol derivative in which two halogen atoms are addedto ⁸C and ¹²C, respectively. The dihalide was prepared similarly. Theprepared dihalide tended to eliminate a hydrogen chloride in anhydrousorganic solvent under basic (alkaline) condition to afford a monohalidein which the double bond between ⁸C and ¹²C was rebuilt.

Halogenation of curcumol ether derivatives and elimination of hydrogenhalide:

Halogenation of curcumol ester derivatives and elimination of hydrogenhalide:

Halogenation of curcumol sulfate derivatives and elimination of hydrogenhalide:

The method for the addition of curcumol to hydrogen halide is similar tothat of the addition of curcumol to halogen, with the exception that Hwas added to ⁸C, while X was added to ¹²C.

The above prepared bromide or chloride was dissolved in anhydrousmethanol. Fluoride (e.g., NH₄F, NaF) was added and reacted at roomtemperature, yielding the corresponding curcumol fluoride.

The method for the preparation of the curcumol ester halide, in which Kcontains a double bond, is as follows: curcumol halide was used as astarting material, and then the curcumol ester halide in which Rcontains a double bond was obtained according to the aboveesterification method of curcumol.

The Replacement of the Halogen Atom in the Curcumol Halide

One of the above prepared derivative (or curcumol) halides (monohalidesor dihalides), mainly bromide, was dissolved in an appropriate anhydrousorganic solvent (acetonitrile, tetrahydrofuran and the like), and anexcess of (generally 2-3.5 folds in mole) dried amine R²—H (herein R² isan amine, including nitrogen-containing heterocycle) was added andreacted at room temperature for more than 3 hours. The product wasseparated by silica gel column or purified by recrystallization toafford the corresponding curcumol amine derivative or the salt thereof(R¹ and R² are defined as above).

Amination of monohalide (The bond between ⁸C and ¹²C is a double bond)(R¹ and R² are defined as above):

Amination of monohalide (The bond between ⁸C and ¹²C is a single bond)(R¹ and R² are defined as above):

Amination of dihalide: The amination of dihalide provided a large amountof monoaminated compound (formula II) in which the bond between ⁸C and¹²C was a double bond and a small amount of diaminated compound (formulaIII)(R¹ and R² are defined as above):

Curcumol (or the above prepared curcumol derivatives) monohalide (thebond between ⁸C and ¹²C is a double bond or a single bond) was dissolvedin an appropriate organic solvent (methanol, ethanol, acetone and thelike), and reacted with NaOH solution at room temperature. The halogenatom was replaced by a hydroxy group (R¹ is defined as above):

The above reaction provided the curcumol ester derivative at ¹²C ofwhich a hydroxy group was added. The hydroxy group at ¹²C also could beesterified or etherified to afford the corresponding curcumolderivatives (R¹ is defined as above):

Curcumol halide (mainly bromide) was dissolved in an appropriate solvent(50%-95% methanol solution in water, and the like), and reacted withsilver nitrate solution to afford the corresponding curcumol nitratederivative.

The Preparation of Curcumol Derivatives in Which the Double Bond isOxidized

The double bond between ⁸C and ¹²C of curcumol could be oxidized by theoxidizing agents such as potassium permanganate, H₂O₂ and the like in anappropriate organic solvent (acetone, acetic acid, and propionic acid,and the like,) to afford curcumol dihydroxy derivative or curzerenonederivative.

Curcumol was oxidized with equal mole of potassium permanganate in theneutral to basic acetone solution to afford curcumol dihydroxyderivative:

Curcumol was oxidized with the excess amount of the solution ofpotassium permanganate in acetic acid to afford the curzerenonederivative:

The derivative produced in the reaction can be esterified or etherifiedto afford the corresponding ester or ether.

The Preparation of Curcumol Epoxy Derivative

Ccurcumol was dissolved in an appropriate organic solvent (chloroform,dichlormethane, and the like), and m-chlorobenzoyl hydroperoxide wasadded at room temperature. Then the mixture was washed with 5% NAOHsolution several times and dried over anhydrous sodium sulfate. Theorganic solvent was removed under vacuum and the residue was separatedby silica gel column to afford the compound of formula (III), wherein Zis

The oxygen ring of the above compound was opened in mild acidic solutionto afford the compound of formula (II) wherein R² is OH and R³ is H. Theesterification (or alkylation) of the ring-opening compound (Method 1:the hydroxy group at 6C of curcumol was esterified; Method 2: thehydroxy group at 6C of curcumol was alkylated) afforded the compound offormula (II) wherein R³ is H and R² is OR¹ (wherein R¹ is defined asabove).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the measured OD450 of HBV e antigen sample upon treatmentwith curcumol and the compounds according to this invention.

FIG. 2 shows the decreased percent ages of HBeAg sample relative to thatof the positive control, upon treatment with curcumol and the compoundsaccording to this invention.

MODE OF CARRYING OUT THE INVENTION

The present invention is further illustrated in combination with thefollowing examples.

Preparation Example 1 Preparing Curcumol Ester (the Hydroxy Group at ⁶C)

(1) 2.48 g (95%, 0.01 mol) of curcumol was dissolved in 50 mL ofchloroform. A small amount of anhydrous magnesium sulfate was added andstirred for 3 hours. The magnesium sulfate was removed by filtration.Then a small amount of triethylamine was added, and 1.06 g of acetylchloride (98%, 0.11 mol) was added dropwise at room temperature. Theresulting mixture was stranded for 24 hours and then poured into crushedice. The solution in chloroform was washed with cold water until thesolution was neutral. The aqueous phase was discarded and the organicphase was dried over anhydrous magnesium sulfate. The chloroform wasremoved under vacuum, to obtain a light yellow product, which wasseparated with 95:5 petroleum ether (60-90° C.): ethyl acetate and200-300 mesh silica gel column to afford 1.55 g curcumol acetate (thehydroxyl group in the parent compound was esterified), i.e. thederivative of Formula (III), wherein, R¹ is CH₃CO (Compound No. 1), as acolourless to white oil. Yield: 58%, HPLC: 99.5%.

Element analysis using Thermo Finnigam Company (U.S.) FlashEA1112element analysis apparatus found: C, 73.554%; H, 9.308%; Molecularformula (C₁₇H₂₆O₃) calculated: C, 73.344%; H, 9.414%.

Finnigan MAT 8430 Mass spectrometric analysis showed that the molecularweight of the product was 278.

The spectrum showed 17 carbon signals, 26 proton signals, usingBrukerACF-300 nuclear magnetic resonance spectrometer and using CDCl₃ asa solvent.

This confirmed that the product was Compound No. 1.

(2) 2.48 g (95%, 0.01 mol) of curcumol was dissolved in 50 mL ofdioxane, and a small amount of anhydrous magnesium sulfate was added andstirred for 3 hours. The magnesium sulfate was removed by filtration. Anappropriate amount of DMAP and DPC were added, and then 0.91 g ofCH₃CH₂COOH (98%, 0.012 mol) was added. The mixture was reacted at 70° C.for 100 hours, and then poured into ice water. The target product in thedecanted solution was extracted several times by solvents such as ethylether or ethyl acetate. The aqueous phase was discarded and the organicphase was combined. The organic solvent was removed after dried overanhydrous magnesium sulfate. The resulting crude product was separatedby silica gel column, yielding 2.15 g of curcumol propionate withrelatively high purity as a colorless oil, i.e. the derivative offormula (III) wherein R¹ is CH₃CH₂CO, yield: 82.1%, HPLC: 98.8%.

Finnigan MAT8430 Mass spectrometric analysis showed that the molecularweight of the product was 292.

The spectrum showed 18 carbon signals, 28 proton signals, usingBrukerACF-300 nuclear magnetic resonance spectrometer and using CDCl₃ asa solvent.

(3) 2.48 g (95%, 0.01 mol) of curcumol was dissolved in 30 mL of aceticanhydride and heated under reflux for several hours. Then the reactionmixture was poured into ice water and the excessive acetic anhydride wasneutralized with aq. NaOH solution. The pH of aqueous solution wasadjusted to 7-8. The target product in the decanted solution wasextracted several times by solvents such as ethyl ether or ethylacetate. The aqueous phase was discarded and the organic phase wascombined. The organic solvent was removed after dried over anhydrousmagnesium sulfate. The resulting crude product was separated by silicagel column, yielding 1.8 g of curcumol propionate with relative highpurity as a light yellow oil, i.e. Compound No. 1, yield 64%. HPLC:99.1%.

(4) 2.48 g (95%, 0.01 mol) of curcumol was dissolved in 50 mL ofchloroform, and 1.9 g (97%, 0.012 mol) of p-methyl benzoyl chloride and5 mL of pyridine were added. The mixture was heated under reflux forseveral hours and poured into ice water. The target product in thedecanted solution was extracted several times by solvents such as ethylether or ethyl acetate. The aqueous phase was discarded and the organicphase was combined. The organic solvent was removed after dried overanhydrous magnesium sulfate. The resulting crude product was separatedby silica gel column, yielding 2.3 g of curcumol p-methyl benzoate withrelative high purity as a white oil, i.e. the derivative of formula (I)wherein R¹ is CH₃ArCO (as shown in the following formula XV), yield64.7%. HPLC: 99.5%.

Finnigan MAT8430 Mass spectrometric analysis showed that the molecularweight of the product was 354.

The spectrum showed 23 carbon signals, 30 proton signals, usingBrukerACF-300 nuclear magnetic resonance spectrometer and using CDCl₃ asa solvent.

(5) 1 g of curcumol was dissolved in 30 mL of DMF, and 2 mL of thionylchloride was dissolved in 10 mL of DMF. The thionyl chloride solutionwas added dropwise to the curcumol solution. The temperature of thereaction solution was kept at 20-25° C. and reacted for 3 hours. Thenthe reaction mixture was poured into 100 mL of water and hydrolyzed for2 hours. The mixture was extracted with 60 mL of ethyl ether, yieldingoily curcumol sulfate, which was separated by silica gel column toafford 0.87 g of product, HPLC: 98.5%. The product was dissolved in 5 mLof methanol and adjusted with 1N NaOH solution to pH 8. The resultingmixture was evaporated to dryness and the residue was treated with ethylacetate, yielding sodium curcumol sulfate salt as a brown powder, whichwas soluble in water, i.e. the derivarive of formula (III) wherein R¹ isHO₃S or NaO₃S (Compound No. 20 or Compound No. 21). The NaO₃S derivativewas detected:

Nuclear magnetic resonance spectrum showed 15 carbon signals, 23 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 354.

Preparation Example 2 Preparing Alkylated Curcumol (the Hydroxy Group at⁶C)

To a 100 mL of three-necked flask equipped with a stirrer, a thermometerof 0-50° C. and a 25 mL of constant pressure dropping funnel were added2.45 g (0.01 mol, 96%) of curcumol, 60 mL of isopropyl ether and 5 mL ofdichlormethane. After complete dissolvation under stirring, a smallamount of anhydrous magnesium sulfate was added and stirred for 3 hours.Magnesium sulfate was removed by filtration. 1.55 g (0.012 mol) ofdimethyl sulfate was dissolved in the constant pressure dropping funnelcontaining 10 mL of dried isopropyl ether. The stirring was initiatedand the temperature of the solution was kept at 35° C. The dimethylsulfate solution was added dropwise slowly. After the completion ofaddition, NaOH solution (0.025 mol) was added dropwise and continued tostir for more than 12 hours. Then the reaction mixture was poured into60 g of ice water and extracted with 70 mL of ethyl ether three times(30, 30, 10 mL). The combined ethyl ether was washed with water until itwas neutral. Then the ethyl ether solution was dried over anhydrousmagnesium sulfate. The magnesium sulfate was removed by filtration. Theethyl ether was removed under vacuum at room temperature to afford apuce solid, which was purified with 1: 50-100 folds of silica gel(200-300 mesh) and eluted with petroleum ether: ethyl acetate=95:5,yielding 1.9 g (content: 99.2% (HPLC)) of the curcumol derivative withthe hydroxy group at ⁶C being methylated, (i.e. the compound of formulaIII wherein R¹ is CH₃—, and R² is H), yield: 76%, MP: 71-73° C.

Element Analysis found: C, 76.935%; H, 10.296%.

Molecular formula (C₁₆H₂₆O₂) calculated: C, 76.752%; H, 10.467%.

Nuclear magnetic resonance spectrum showed 16 carbon signals, 26 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 250.

This indicated that the resulting product was our tardet product, i.e.the curcumol methyl ether derivative with ⁶C being converted intomethoxy. The formula was as follows:

-   -   curcumol methyl ether derivative sodium curcumol sulfate

Preparation Example 3 Preparing the Adduct Obtained from Curcumol andHalogen

(1) 2.45 g (0.01 mol, 96%) of curcumol was dissolved in 45 mL ofmethanol. The mixture was dried over a small amount of anhydrousmagnesium sulfate, and then the magnesium sulfate was removed byfiltration. The reactant was kept at 5-10° C. and 0.61 mL of (3.15 g/mL,0.012 mol) of dry liquid bromine was added dropwise to the solution ofcurcumol in methanol slowly under good stirring. After the addition ofbromine, the reaction was continued to stir for 5-30 minutes until thesolution was colorless. The methanol was removed under vacuum, yieldingcrude curcumol bromide, which was directly used in Example 6, or thislight yellow crude product may be separated by silica gel column toafford 2.81 g of relatively pure target product as a nearly colorlessthick oil, yield: 71%, HPLC: 99.2%, i.e. the curcumol dibromide offormula II wherein R¹ is H, R², R³ are both bromine atom (Compound No.30).

Element Analysis found: C, 45.630%; H, 6.028%.

Molecular formula (C₁₅H₂₄O₂Br₂) calculated: C, 45.477%; H, 6.107%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 24 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 396.

This indicated that the resulting product was our target product,Compound No. 30.

(2) 2 g (0.005 mol) of dibromide from step (1) was dissolved in 40 mL ofanhydrous methanol. A small amount of anhydrous magnesium sulfate wasadded and stirred. The magnesium sulfate was removed by filtration, andthen a dried solution of 0.21 g (95%, 0.005 mol) NaOH in methanol wasadded. The mixture was stranded for more than 12 hours at roomtemperature. Then the dark reaction mixture was poured into water andextracted with ethyl ether several times. The ethyl ether layer waswashed with water until the pH of the wash solution was about 7. Afterthe ethyl ether was dried over anhydrous magnesium sulfate, the solventwas removed under vacuum to afford a dark and thick substance. Afterseparating by silica gel column, a curcumol derivative with removal of ahydrogen bromide between the carbon carbon atoms of 8- and 12-positionand thus formation of a double bond was obtained. 0.83 g of curcumolmonobromide derivative (Compound No. 34) was obtained, yield: 53%, HPLC:98.2%, a light yellow oil (Formula XI, wherein R¹ is H, R² is Br).

Element Analysis found: C, 57.214%; H, 7.650%.

Molecular formula (C₁₅H₂₄O₂Br) calculated: C, 56.967%; H, 7.649%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 24 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 316.

This confirmed that the obtained product was Compound No. 34.

Preparation Example 4 Preparing the Adduct Obtained from Curcumol andHalogen Acid

2.45 g (0.01 mol, 96%) of curcumol was dissolved in 45 mL of methanol. Asmall amount of anhydrous magnesium sulfate was added and stirred, andthen the magnesium sulfate was removed by filtration. The reactant waskept at 5-10° C. and an excess of dry hydrogen bromide (the mole ofhydrogen bromide is 1.2 fold of the mole of curcumol) was slowlyintroduced to the solution of curcumol in methanol slowly under goodstirring. After this addition, the reaction mixture was continued tostir about 1 hours, yielding puce crude curcumol monobromide, which wasseparated by silica gel column to afford relatively pure target productcurcumol monobromide, i.e., the curcumol monohalide of formula (II),wherein R³ is bromine atom and R² is H (Compound No. 34).

Element Analysis found: C, 57.965%; H, 7.582%.

Molecular formula (C₁₅H₂₅O₂Br) calculated: C, 56.786%; H, 7.943%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 25 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 317.

This indicated that the obtained product was our target compound.

The bromide or fluoride obtained above was dissolved in anhydrousmethanol, and to it was added fluoride (such as NH₄F, NaF) and reactedat room temperature, yielding the corresponding curcumol fluoride.

Preparation Example 5 Preparing of Hydrogenated Curcumol

(1) 2.45 g (0.01 mol, 96%) of curcumol was dissolved in 45 mL ofmethanol. A small amount of anhydrous magnesium sulfate was added andstirred, and then the magnesium sulfate was removed by filtration. Asmall amount of catalyst for the hydrogenation (such as Pd/C and thelike) was added, and hydrogen was introduced under room temperature andnormal pressure with vigorous stirring. The hydrogenation reaction waskept for more than 24 hours. After the completion of the hydrogenationreaction, Pd/C was recovered by filtration. The solvent methanol of thefiltrate was removed under vacuum at room temperature to afford a lightyellow oil. After purified by silica gel column, 1.9 g of nearlycolorless powder was obtained with the double bond at position 8-12 ofcurcumol addition by hydrogen (curcumol dihydrogen derivative), HPLC:98.1%, Melting point: 75-77° C., i.e., the curcumol dihydrogenderivative of formula (II), wherein R¹ is H, R², R³ are both H (CompoundNo. 33).

Element Analysis found: C, 75.832%; H, 10.856%.

Molecular formula (C₁₅H₂₆O₂) calculated: C, 75.581%; H, 10.994%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 26 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 238. This indicated that the obtained product was our targetcompound.

(2) 1 g (96%, 0.004 mol) of curcumol was dissolved in 50 mL of anhydrousethyl ether. To the reaction mixture was added dropwise LiAlH₄ (about0.005 mol) at room temperature. After addition, the mixture wascontinued to stir for 3 hours under dry condition. The ethyl ether wasremoved under vacuum to afford a light yellow powder. After purified bycolumn, 0.58 g of white powder was obtained, yield: 61%, HPLC: 98%,Melting point: 78-80° C., i.e. curcumol derivative of formula (II),wherein R², R³ are both H (Compound No. 33) was obtained.

Element Analysis found: C, 75.832%; H, 10.856%.

Molecular formula (C₁₅H₂₆O₂) calculated: C, 75.581%; H, 10.994%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 26 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 238.

This indicated that the obtained product was our target compound,dihydro curcumol derivative (Compound No. 33).

Preparation Example 6 Preparation of Halogen Atom Replacement Product ofCurcumol Halide

(1) 6.0 g (66%, 0.01 mol) curcumolbromide prepared in PreparationExample 3(1) was dissolved in 80 mL of acetonitrile. A small amount ofanhydrous magnesium sulfate was added and stirred for 3 hours, and thenthe magnesium sulfate was removed by filtration. 2.6 g (98%, 0.033 mol)of dried n-butyl amine was added and stranded for more than 24 hours atroom temperature. The solvent was removed under vacuum at roomtemperature. The light yellow mixture was dissolved in 50 mL of waterand 60 mL of ethyl acetate and was placed in a pear shape separatoryfunnel. The solution was adjusted to pH more than 9 with alkalinesolution such as ammonia and was shaken thoroughly. The aqueous layerwas extracted with 10 mL of ethyl acetate once. The ethyl acetate layerwas combined and washed with water (20 mL×4). 40 mL of water was added,and the solution was adjusted to pH less than 7, preferably pH 3 withhydrochloric acid and shaken thoroughly. The ethyl acetate wasdiscarded. The acidic aqueous solution was washed with 20 mL of ethylether (three times), and the ethyl ether was discarded. 60 mL of newethyl acetate was mixed with aqueous phase, and the mixture was adjustedto pH>9 and shaken thoroughly. The aqueous layer was extracted with 10mL of ethyl acetate once. The ethyl acetate was combined and washed withwater (20 mL×4), and then dried over anhydrous magnesium sulfate. Theethyl acetate was removed under vacuum at room temperature to afford 3.2g of light colored crystal. After separated by column, 2.2 g of thecorresponding curcumol n-butyl amine compound was obtained, (orrecrystallized with acetone) to obtain a colorless granule crystal,yield: 71.66%, HPLC: 99.8%. Melting point: 104-106° C., i.e., thederivative of formula (III), wherein R¹ is H, R² is CH₃CH₂CH₂CH₂NH(Compound No. 13).

Element Analysis found: C, 74.402%; H, 10.650%; N, 4.62%.

Molecular formula (C₁₉H₃₃O₂N) calculated: C, 74.267%; H, 10.749%; N,4.56%.

Nuclear magnetic resonance spectrum showed 19 carbon signals, 33 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 307.

This indicated that the obtained product was Compound No. 13.

The curcumol n-butyl amine derivative prepared above was dissolved inacetone. Concentrated hydrochloric acid was added dropwise (or HCl gaswas introduced) at 20-30° C. with stirring. The solution was made pH6-7. White curcumol n-butyl amine hydrochloride solid precipitated outof the solution immediately. The solid was filted, washed and dried toafford curcumol n-butyl amine derivative hydrochloride crystai of highpurity (Compound No. 14). HPLC: 99.95%, Melting point: 127-129° C.

(2) 6.0 g (66%, 0.01 mol) curcumol bromide prepared in PreparationExample 3(1) was dissolved in 80 mL of acetonitrile. A small amount ofanhydrous magnesium sulfate was added and stirred for 3 hours, and thenthe magnesium sulfate was removed by filtration. 2.6 g (98%, 0.033 mol)of dried t-butyl amine was added and stranded for more than 24 hours atroom temperature. The solvent was removed under vacuum at roomtemperature. The light yellow mixture was dissolved in 50 mL of waterand 60 mL of ethyl acetate and was placed in a pear shape separatoryfunnel. The solution was adjusted to pH more than 9 with alkalinesolution such as ammonia and was shaken thoroughly. The aqueous layerwas extracted with 10 mL of ethyl acetate once. The ethyl acetate layerwas combined and washed with water (20 mL×4). 40 mL of water was added,and the solution was adjusted to pH<7, preferably pH 3 with hydrochloricacid and shaken thoroughly. The ethyl acetate was discarded. The acidicaqueous solution was washed with 20 mL of ethyl ether (three times), andthe ethyl ether was discarded. 60 mL of new ethyl acetate was mixed withaqueous phase, and the mixture was adjusted to pH>9 and shakenthoroughly. The aqueous layer was extracted with 10 mL of ethyl acetateonce. The ethyl acetate was combined and washed with water (20 mL×4),and then dried over anhydrous magnesium sulfate. The ethyl acetate wasremoved under vacuum at room temperature to afford 3.0 g of lightcolored crystal. After separated by silica gel column, 2.0 g of thecorresponding curcumol t-butyl amine compound was obtained as acolorless solid (Compound No. 15), yield: 65.4%, HPLC: 98.8%.

Melting point: 79-80° C. i.e. the derivative of formula (III) wherein R¹is H, R² is (CH₃)₃CNH—.

Nuclear magnetic resonance spectrum showed 19 carbon signals, 33 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 307.

This indicated that the obtained products were our target compounds withthe following formula:

The curcumol t-butyl amine derivative prepared above was dissolved inacetone. Concentrated hydrochloric acid was added dropwise (or HCl gaswas introduced) at 20-30° C. with stirring. The solution was made pH6-7. White curcumol t-butyl amine hydrochloride powder solidprecipitated out when acetone was evaporated (Compound No. 16). Thestructure formula is shown above. HPLC: 99.95%, Melting point: 115-116°C.

(3) 6.0 g (66%, 0.01 mol) curcumol bromide prepared in PreparationExample 3(1) was dissolved in 80 mL of acetonitrile. A small amount ofanhydrous magnesium sulfate was added and stirred for 3 hours, and thenthe magnesium sulfate was removed by filtration. 3.34 g (98%, 0.03 mol)of p-hydroxy aniline was added and stranded for more than 24 hours atroom temperature. The solvent was removed under vacuum at roomtemperature. The dark mixture was dissolved in 50 mL of water and 100 mLof ethyl acetate and was placed in a pear shape separatory funnel. Thesolution was adjusted to pH>9 with alkaline solution such as ammonia andwas shaken thoroughly. The aqueous layer was extracted with 10 mL ofethyl acetate once. The ethyl acetate layer was combined and washed withwater of pH 8-9 (20 mL×4). Then 40 mL of water was added, and thesolution was adjusted to pH<7, preferably pH 3 with hydrochloric acidand shaken thoroughly. The ethyl acetate was discarded. The acidicaqueous solution was washed with 20 mL of ethyl ether (three times), andthe ethyl ether was discarded. 100 mL of new ethyl acetate was mixedwith aqueous phase, and the mixture was adjusted to pH>8-9 and shakenthoroughly. The aqueous layer was extracted with 10 mL of ethyl acetateonce. The ethyl acetate was combined and washed with water (20 mL×4),and then dried over anhydrous magnesium sulfate. The ethyl acetate wasremoved under vacuum at room temperature to afford 3.8 g of light yellowsolid. After separated by silica gel column, 2.30 g of the correspondingcurcumol p-hydroxy aniline compound was obtained, (or afterrecrystallization) to obtain a white crystal, yield: 67.1%, HPLC: 99.2%,i.e. the derivative of formula (III) wherein R¹ is H, R² is HOArNH—.(Compound No. 5). The structure formula was shown below:

The p-hydroxy aniline derivative of curcumol prepared above wasdissolved in acetone. Concentrated hydrochloric acid was added dropwise(or HCl gas was introduced) at 40-50° C. with stirring. The solution wasmade pH 5-6. A light yellow curcumol amine hydrochloride crystalprecipitated out after being stranded, i.e. off-white curcumol p-hydroxyaniline hydrochloride crystal of high purity was obtained (Compound No.6, the structure formula is shown above). HPLC: 99.89%, Melting point:147.5-149.5° C. The hydrochloride was detected:

Nuclear magnetic resonance spectrum showed 21 carbon signals, 30 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 379.5.

This indicated that the obtained product was our target compound.

(4) 6.0 g (66%, 0.01 mol) curcumol bromide prepared in PreparationExample 3(1) was dissolved in 80 mL of acetonitrile. A small amount ofanhydrous magnesium sulfate was added and stirred for 3 hours, and thenthe magnesium sulfate was removed by filtration. 2.90 g (98%, 0.031 mol)of anhydrous piperazine was added and stranded for more than 24 hours atroom temperature. The solvent was removed under vacuum at roomtemperature. The light yellow mixture was dissolved in 50 mL of waterand 100 mL of ethyl acetate and was placed in a pear shape separatoryfunnel. The solution was adjusted to pH>9 with alkaline solution such asammonia and was shaken thoroughly. The aqueous layer was extracted with10 mL of ethyl acetate once. The ethyl acetate was combined and washedwith water of pH 8-9 (20 mL×4). Then 40 mL of water was added, and thesolution was adjusted to pH 3 with hydrochloric acid. The ethyl acetatewas discarded. The acidic aqueous solution was washed with mL of ethylether (three times), and the ethyl ether was discarded. 100 mL of newethyl acetate was mixed with acidic aqueous phase, and the mixture wasadjusted to pH>9 and shaken thoroughly. The aqueous layer was furtherextracted with 10 mL of ethyl acetate once. The ethyl acetate wascombined and washed with water (20 mL×4), and then dried over anhydrousmagnesium sulfate. The ethyl acetate was removed under vacuum at roomtemperature to afford 3.1 g of white solid. After separated by silicagel column, 2.3 g of the corresponding curcumol piperazine derivativewas obtained, (or after recrystallization) to obtain a white crystal,yield: 71.8%, HPLC: 99.2%, Melting point: 128-130° C., i.e. thederivative of formula (III) wherein R¹ is H, R² is

Nuclear magnetic resonance spectrum showed 19 carbon signals, 32 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 320.

This confirmed that the obtained product was Compound No. 7.

The above-prepared piperazine derivative of curcumol was dissolved inacetone. Concentrated hydrochloric acid was added dropwise (or HCl gaswas introduced) at 40-50° C. with stirring. The solution was made pH5-6. A white curcumol amine hydrochloride crystal precipitated out afterstranded. The crystal was decolored by activated carbon in water andrecrystallized, yielding off-white curcumol piperazine hydrochloridecrystal of high purity (Compound No. 8). HPLC: 99.89%, Melting point:150-152° C.

Similarly, using the above method, ethyl amine was reacted with curcumoldihalide, yielding the curcumol ethyl amine derivative as a beige powdercrystal, i.e. the derivative of formula (III) wherein R¹ is H, R² isCH₃CH₂NH, Melting point: 92-94° C., yield: 76%.

(5) 6.0 g (66%, 0.01 mol) curcumol bromide prepared in PreparationExample 3(1) was dissolved in 40 mL of the solution of methanol in water(methanol: water=90:10). 1.5 g of 30% NaOH solution was added dropwiseover 10 hours at room temperature. The reaction mixture was stranded formore than 24 hours. The pH of solution was adjusted to neutral. Then 40mL of water was added and the mixture was extracted with 50 mL of ethylacetate several times. The ethyl ether was combined and washed withwater several times, and then dried over a small amount of anhydroussodium sulfate. The solvent was removed under vacuum to afford 1.4 g ofpuce and thick oil. After separated by silica gel column, 0.87 g ofcurcumol dihydroxy derivative was obtained as a light colored crystal,yield; 65%, Melting point: 163-165° C., HPLC: 98.3%, i.e. the compoundof formula (II) wherein R³, R² are both OH (Compound No. 32, curcumoldihydroxy compound).

Element Analysis found: C, 66.873%; H, 9.458%.

Molecular formula (C₁₅H₂₆O₄) calculated: C, 66.636%; H, 9.693%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 26 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 270.

This indicated that obtained the products were our target compounds withthe following formula:

(6) 6.0 g (66%, 0.01 mol) curcumol bromide prepared in PreparationExample 3(1) was dissolved in 60 mL of methanol. To this solution wasadded dropwise 10 mL of aqueous solution containing 3.6 g (99%, 0.02mol) silver nitrate and stranded overnight. A large amount of whiteflocculent precipitate separated out. 60 mL of water and ether (1:1) wasadded and extracted twice. Silver bromide was recovered from aqueousphase, and ethyl ether phase was washed with 40 mL of water twice, anddried over anhydrous sodium sulfate. The solvent was removed undervacuum to afford curcumol dinitrate derivative, i.e. the derivative offormula (II), wherein R³, R² are replaced by NO₃. The structure formulawas shown above (Compound No. 31, curcumol dinitrate derivative).

Preparation Example 7 Preparing Curcumol Derivative with Double Bondbeing Oxidized

(1) 2.45 g (0.01 mol. 96%) of curcumol was dissolved in 40 mL ofacetonitrile. The resulting mixture was treated with an aqueous solutioncontaining 1.6 g potassium permanganate (98%, 0.01 mol) and stranded atroom temperature until the amaranth of the solution was completelyfaded. Then the acetonitrile was removed under vacuum. The residue wasextracted with 1:1 ethyl acetate-water (100 mL). The aqueous phase wasdiscarded and the ethyl acetate phase was washed with water twice, andthen dried over anhydrous sodium sulfate. A dark solid was obtainedafter the solvent was removed. After separated by silica gel column, 2.1g of off-white crystal was obtained, yield, 77.7%, Melting point:163-165° C., HPLC: 98.8%, i.e. the compound of formula (II) wherein R¹is H, R² and R³ are OH (curcumol dihydroxy compound), whose solubilityin water is very big.

Element Analysis found: C, 66.873%; H, 9.458%.

Molecular formula (C₁₅H₂₆O₄) calculated: C, 66.636%; H, 9.693%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 26 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 270.

This indicated that the obtained product was our target compound.

The resulting curcumol dihydroxy derivative was alkylated or esterifiedto afford the curcumol derivative of formula (II) wherein R⁵, R⁶ areboth R¹O, R¹ was defined as above.

(2) 2.45 g (0.01 mol, 96%) of curcumol was dissolved in 40 mL of aceticacid, and to it was added an aqueous solution dissolved with 4 gpotassium permanganate. The mixture was heated under reflux for severalhours followed by removal of the organic solvent under vacuum. Themixture was extracted with 100 mL of 50% ethyl acetate and water. Theethyl acetate was washed with water several times and dried overanhydrous sodium sulfate. The solvent wad removed under vacuum to afforda dark solid. After separated by silica gel column, 1.45 g of whitecurzerenone derivative was obtained, yield: 61%, HPLC: 98%, Meltingpoint: 138-140° C., i.e. Compound No. 31 of formula (III) wherein Z isO.

Element Analysis found: C, 70.865%; H, 9.102%.

Molecular formula (C₁₄H₂₂O₃) calculated: C, 70.560%; H, 9.305%.

Nuclear magnetic resonance spectrum showed 14 carbon signals, 22 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 238.

This indicated that the obtained product was our target compound withthe following formula.

Preparation Example 8 Preparing the Epoxy Derivative of Curcumol

1 g curcumol (96%, 0.004 mol) was dissolved in 100 mL of chloroform. Toit was added 2 g m-chloro-benzoyl hydroperoxide (98%, 0.011 mol) at roomtemperature, and reacted at 15-20° C. for 8 hours. The mixture waswashed with 5% sodium hydroxide solution several times and furtherwashed with water to be neutral, and then dried over anhydrous sodiumsulfate. The solvent was removed under vacuum. The residue was purifiedby silica gel column, to afford the epoxy derivative of curcumol offormula (III)(Compound No. 48), wherein, Z is

yielding 0.504 g white powder, yield: 50%, HPLC: 98.5%. Melting point:130-132° C.

Element Analysis found: C, 70.865%; H, 9.102%.

Molecular formula (C₁₅H₂₄O₃) calculated: C, 71.035%; H, 9.046%.

Nuclear magnetic resonance spectrum showed 15 carbon signals, 24 protonsignals, using CDCl₃ as a solvent.

Mass spectrometric analysis showed that the molecular weight of theproduct was 252.

This indicated that the obtained product was our target compound withthe following formula.

The oxygen ring of the above compound was opened in mild acidic solutionto afford the compound of formula (II) wherein R² is OH, R³ is H. Theesterification (or alkylation) of the ring-opening compound (Method 1:the hydroxy group at ⁶C of curcumol was esterified; Method 2: thehydroxy group at 6C of curcumol was alkylated) afforded the compound offormula (II) wherein R³ is H, R² is OR¹ (wherein R¹ was defined asabove).

TEST EXAMPLES Example 1 Measurement of In Vitro Inhibition Rate to TumorCells

1. Experimental Compounds

Fifty compounds in total: new compounds 1-49, and Curcumol E.

2. Materials

(1) MTT MTT was dissolved in 0.01 mol/L phosphate buffered saline (PBS)at a final concentration of 4 mg/ml. The resulting solution wassterilized by filtration, and stored at 4° C.

(2) Preparation of MTT lysis solution 80 g of sodium dodecylsulphate wasdissolved in 200 ml of DMF with water bath. 200 ml of distilled waterwas added. The pH value was adjusted to 4.7 with 80% acetic acid and 1NHCl (1:1).

(3) Tumor cell lines

U-937 human histocytic lymphoma A-549 human lung adenocarcinoma Bel-7402human hepatic carcinoma MCF-7 human mammary adenocarcinoma Hela humancervical carcinoma HL-60 human promyelocytic leukemia SMMC-7721 humanhepatic carcinoma LLC mouse Lewis lung carcinoma

3. Methods

(1) Single cell suspension was seeded into 96-well plates (×3) (thecells was diluted in the PRMI-1640 minimum medium to 3×10⁴ cells/ml, 200ug diluted cells/well). The cells were incubated in an atmosphere of 5%CO₂, 100% humidity for 24 hours at 37° C. The experiments were performedin quadruplicate.

(2) The medium was removed. Solutions of the anti-tumor agents (15compounds) were prepared in a freshly prepared medium at theconcentrations indicated in the following Table. 200 μg of the solutionwas added into each well. Incubation was continued for 48 hours.

(3) 20 μg of 2 mg/ml MTT was added into each well, and incubated for 4hours.

(4) The medium was removed from the well. The microplates were swiftlyturned down to throw-off the medium. 150 μg/well DMSO was added intoeach well, and vortexed at room temperature for 10 minutes.

(5) OD value (λ=570 nm) was measured with an Enzyme-Linked Detector.

(6) The inhibition rates (IR) of cell growth were respectivelycalculated based on the following equation:IR(%)=[1−(average OD of the experimental group/average OD of the controlgroup)]×100%

The results are shown in Table 1. The results indicate that theinhibition rate to Hela cells of all of the compounds exhibited>50%.

The experiments were repeated three times. The results are shown inTables 1, 2 and 3.

TABLE 1 Results of the screening test for in vitro anti-tumorbioactivity of the compounds Testing parameter: inhibition rateConcentration: 50 μg/ml Results Results Results Results Results ResultsResults (%) Results (%) (%) (%) (%) (%) (%) (%) No. U-937 A-549 Bel-7402MCF-7 SMMC-7721 HL-60 Hela LLC  1 45.45 −9.16 32.12 45.6 23.10 23.1251.23 1.30  2 71.17 4.93 23.33 45.98 12.35 12.34 70.56 2.98  3 91.727.76 14.41 32.15 8.02 3.21 69.23 6.89  4 52.23 10.51 43.21 49.23 5.3042.1 73.22 1.01  5 93.95 90.32 80.21 90.32 85.01 91.32 94.36 3.21  690.92 91.84 81.84 91.89 86.12 93.21 95.26 88.23  7 93.01 65.32 55.4488.32 3.34 90.56 90.38 85.42  8 90.26 60.5 54.32 89.12 4.50 91.95 91.8712.1  9 87.32 65.21 45.32 56.47 1.21 86.32 82.36 4.83 10 85.20 62.3144.68 56.32 7.79 87.20 87.98 4.18 11 85.3 60.12 43.25 58.39 33.88 70.2391.64 11.77 12 80.3 60.32 43.08 54.89 32.03 60.21 83.78 −10.89 13 51.107.59 33.16 54.67 3.71 0.12 66.32 0.24 14 82.88 8.09 32.00 56.30 6.142.45 75.46 −10.57 15 57.32 17.43 32.98 65.32 −2.50 8.23 70.02 −11.83 1682.56 6.01 1.23 54.89 −2.50 2.31 68.99 1.13 17 87.18 4.68 −12.36 53.8910.16 92.33 91.58 7.27 18 70.30 −2.24 13.58 57.89 13.39 4.26 60.32 4.1819 50.97 2.51 4.32 12.6 23.01 54.03 55.67 −15.52 20 89.23 12.50 7.8918.9 18.08 91.35 94.23 8.97 21 92.04 26.42 5.36 87.56 −7.26 93.12 91.6910.47 22 60.32 19.23 12.80 88.32 44.39 21.01 63.20 −6.01 23 81.60 −7.4113.54 32.56 18.69 12.50 55.33 12.1 24 63.78 3.12 1.23 8.26 −1.59 2.6354.62 4.83 25 86.94 13.68 5.68 9.53 0.24 −2.63 81.88 4.18 26 55.32 −3.243.45 43.21 0.97 −0.23 51.00 11.77 27 52.98 15.01 4.65 23.78 25.94 3.1962.31 −10.89 28 59.32 1.23 −2.24 32.15 −3.78 2.98 58.75 0.24 29 25.323.89 2.51 37.85 4.26 42.12 56.98 −10.12 30 13.25 4.36 12.40 36.15 1.0313.20 53.87 −1.83 31 −0.68 8.65 0.23 16.75 −1.96 16.50 57.98 1.13 3265.32 −12.5 4.23 65.12 79.96 14.00 84.33 7.27 33 44.34 16.32 14.32 45.3−7.44 1.32 60.89 4.18 34 35.23 2.31 1.11 1.23 −5.25 2.96 50.64 −18.52 357.36 3.89 3.65 3.89 5.23 3.12 59.88 8.97 36 6.98 9.21 0.22 −1.23 15.391.03 62.13 11.56 37 23.56 32.03 15.32 23.10 −2.32 56.23 63.75 1.21 3819.30 3.71 13.56 11.32 −8.7 12.35 58.69 10.47 39 12.68 6.14 3.87 12.84−0.33 3.16 58.75 −3.24 40 4.36 −2.50 13.2 27.35 11.65 8.88 64.26 4.21 418.65 −2.50 16.78 36.45 4.42 6.78 50.32 30.96 42 −12.5 10.16 2.15 34.216.86 2.73 55.64 9.99 43 16.32 13.39 9.15 43.21 −10.12 2.56 62.13 −2.0644 35.64 23.01 −6.54 25.55 −10.53 3.71 55.14 23.35 45 32.19 2.10 3.0126.78 −10.73 6.14 59.45 2.74 46 16.32 3.12 4.55 19.88 −0.86 −2.60 53.12−12.99 47 86.47 −12.0 2.88 65.12 −1.43 10.32 83.66 −13.24 48 90.92 7.8416.40 61.23 20.59 60.80 86.77 −8.04 49 78.65 5.36 2.69 0.65 −11.3 55.3579.8 −0.15 E 28.01 −0.57 4.79 55.64 −11.5 1.32 86.75 −3.04

TABLE 2 (repeated screening). Results of the screening test for in vitroanti-tumor bioactivity of the compounds Testing parameter: inhibitionrate concentration: 50 μg/ml Results Results Results Results ResultsResults Results Results (%) (%) (%) (%) (%) (%) (%) (%) No. U-937 A-549Bel-7402 MCF-7 SMMC-7721 HL-60 Hela LLC  1 34.26 −6.32 25.32 41.02 28.2312.03 55.98 1.30  2 70.30 9.23 12.78 41.78 14.67 17.98 75.23 0.32  393.01 10.32 11.00 22.35 8.96 4.89 70.23 0.02  4 51.00 14.03 43.02 50.091.96 40.01 70.12 0.23  5 95.56 90.00 81.54 91.02 86.91 90.89 92.31 85.07 6 94.12 92.84 81.84 91.89 86.12 93.21 95.26 87.02  7 90.00 67.23 60.2385.01 1.23 91.02 89.30 −2.99  8 91.32 58.56 50.45 85.23 2.30 90.17 92.001.02  9 71.32 60.45 24.32 54.23 11.23 85.02 80.23 2.31 10 80.20 55.0640.32 53.26 4.44 85.03 86.49 2.36 11 85.01 56.32 43.25 57.39 22.13 68.2389.02 1.23 12 80.23 65.01 38.02 55.01 32.09 61.20 85.36 −1.23 13 45.103.56 30.25 50.36 0.23 0.79 65.92 1.32 14 81.23 9.23 23.56 54.23 3.214.35 76.32 −14.29 15 50.12 12.33 31.20 60.12 −8.60 2.98 71.42 −10.12 1680.12 10.19 8.99 50.32 2.63 15.00 70.32 0.22 17 85.12 5.23 −10.90 50.320.32 90.12 92.36 2.31 18 69.23 −0.32 11.23 54.32 0.69 7.32 65.03 0.22 1942.23 3.10 8.95 0.16 12.301 55.98 50.27 −16.00 20 90.32 13.00 5.36 16.321.23 92.37 90.56 9.37 21 89.46 20.01 2.13 85.23 −1.82 90.89 90.11 1.8922 50.23 12.36 0.23 85.02 40.32 21.03 61.88 −6.41 23 83.00 −1.89 10.7723.66 4.96 13.56 60.23 13.20 24 52.31 0.32 4.32 35.02 12.30 0.98 50.121.32 25 82.36 14.23 2.03 8.12 8.32 1.23 75.55 1.02 26 54.23 −3.12 −3.4541.21 −0.97 −0.23 56.55 −11.77 27 50.98 5.23 2.31 14.36 20.36 5.96960.21 −10.00 28 51.23 2.31 −0.45 19.98 −1.02 4.32 60.45 1.23 29 12.321.23 3.21 30.12 5.68 42.79 50.17 −1.95 30 13.10 4.01 9.84 30.56 4.564.12 56.89 −4.65 31 −12.61 4.82 8.42 14.87 −4.56 2.78 55.49 1.25 3267.23 −12.5 4.23 65.02 82.59 13.41 80.12 7.05 33 43.21 14.32 14.89 39.30−1.56 7.68 59.80 3.12 34 23.56 9.23 0.59 4.56 0.95 3.14 51.89 −11.22 350.23 2.77 6.78 9.87 4.32 3.99 64.01 8.23 36 5.32 1.20 0.02 −2.31 14.082.01 65.23 11.56 37 32.10 34.01 12.31 5.61 −8.36 50.12 62.17 0.32 3813.00 0.96 −9.78 10.98 1.23 11.45 60.11 1.96 39 10.32 321 13.56 19.73−0.98 9.58 60.99 −4.57 40 1.02 −12.37 25.43 14.38 −11.36 12.96 64.121.95 41 −1.02 −7.89 1.23 31.28 3.12 1.86 51.23 23.65 42 −1.03 3.12 14.3630.12 0.79 3.14 54.03 1.79 43 12.30 11.24 −4.98 41.94 −8.98 2.00 59.66−11.23 44 23.56 20.33 0.23 24.11 −7.89 5.37 50.01 17.565 45 12.32 0.325.02 20.73 −11.23 1.23 53.12 1.32 46 8.65 3.78 4.65 14.98 −0.45 −4.5649.56 −17.56 47 85.39 −7.85 2.35 60.49 −3.25 1.96 80.79 −14.00 48 91.239.48 10.23 62.38 12.11 50.23 80.79 −4.32 49 75.23 4.23 14.02 1.56 −1.7857.89 75.65 0.12 E 14.321 −11.32 9.87 50.23 −12.30 1.23 70.12 −8.90

TABLE 3 (repeated screening). Results of the screening test for in vitroanti-tumor bioactivity of the compounds Testing parameter: inhibitionrate concentration: 50 μg/ml Results Results Results Results ResultsResults Results Results (%) (%) (%) (%) (%) (%) (%) (%) No. U-937 A-549Bel-7402 MCF-7 SMMC-7721 HL-60 Hela LLC 5 91.03 91.23 78.97 95.53 85.0195.14 94.55 86.54 6 94.09 90.12 83.56 94.13 87.12 95.55 92.33 85.02 791.23 65.29 65.23 84.12 1.23 90.23 89.12 −3.66 8 94.36 55.89 54.89 86.237.98 91.08 91.47 3.12 20  89.98 14.32 1.23 14.23 6.56 91.72 92.47 15.3221  91.45 1.23 3.12 87.58 −2.93 89.79 91.67 7.58 E 19.78 −16.56 9.7851.23 −0.23 1.79 78.56 −14.44

Example 2 Measurement of In Vitro Anti-Tumor IC₅₀ for the Compounds

1. Experimental Compounds

Fifteen compounds in total: 5-14, 17, 20, 21 and E, 5-FU as positivecontrol.

2. Materials

(1) MTT: MTT was dissolved in 0.01 mol/L phosphate buffered saline (PBS)at a final concentration of 4 mg/ml. The resulting solution wassterilized by filtration, and stored in the refrigeratory of 4° C.

(2) Preparing MTT lysis solution: 80 g of sodium dodecylsulphate wasdissolved in 200 ml of DMF with warm water bath. 200 ml of distilledwater was added. The pH value was adjusted to 4.7 with 80% acetic acidand 1N HCl (1:1).

(3) Tumor cell lines

U-937 human histocytic lymphoma A-549 human lung adenocarcinoma Bel-7402human hepatic carcinoma MCF-7 human mammary adenocarcinoma Hela humancervical carcinoma HL-60 human promyelocytic leukemia SMMC-7721 humanhepatic carcinoma LLC mouse Lewis lung carcinoma

3. Methods

(1) Single cell suspension was seeded into 96-well plates (the cells wasdiluted in the PRMI-1640 minimum medium to 3×10⁴ cells/ml, 200 μgdiluted cells/well). The cells were incubated in an atmosphere of 5%CO₂, 100% humidity for 24 hours at 37° C. The experiments were performedin quadruplicate.

(2) The medium was removed. Solutions of the anti-tumor agents (50compounds) were prepared in a freshly prepared medium at serialconcentrations. 200 μg of the solution was added into each well.Incubation was continued for 48 hours.

(3) 20 ug of 2 mg/ml MTT was added into each well, and incubated for 4hours.

(4) The medium was removed from the well. The microplates were swiftlyturned down to throw-off the medium. 150 μg/well of DMSO was added intoeach well, and vortexed at room temperature for 10 minutes.

(5) OD value (λ=570 nm) was measured with an Enzyme-Linked Detector.

(6) The graph of cell vitality was plotted, and IC₅₀ was calculated.

Results

The results are shown in the following Table 4. These resultsdemonstrate that all of the compounds had a comparable or lower IC₅₀against Hela and U-937 cells relative to Compound E and the positivecontrol(5-FU). It indicates that many new compounds had strongeractivities as compared with the parent compound Curcumol, and 5-FU(positive control). Compound 5 and Compound 6 had a strong inhibitoryeffect to all the eight selected tumor cell lines, indicating they areanti-tumor compounds which have a wide-spectrum. Compound 7 and Compound8 showed excellent activity not only to Hela and U-937 cell lines, butalso to U-937 and HL-60 cell lines.

TABLE 4 Results of screening test for bioactivity of the pharmaceuticalcompounds (IC₅₀) IC₅₀ (ug/ml) against tumor cell lines Bel- SMMC- No.Hela U-937 MCF A-549 7402 HL-60 LLC 7721  5 0.32 0.42 1.00 2.0 0.9 0.4 3.6 4.3  6 0.35 0.56 1.10 2.1 1.0 0.51 3.8 4.0  7 0.6 0.76 3.20 1.61 — 8 0.59 0.81 3.28 1.68 —  9 1.5 1.3 10 1.53 2.1 11 2.5 0.5 12 2.4 0.6513 1.6 0.98 14 1.5 0.58 17 1.5 0.92 4.63 1.85 — 20 0.89 0.95 4.98 0.45 —21 0.75 0.89 4.99 — — 1.3  — — E 3.23 — — — — — — — 5-FU 2.86 1.53 0.7 0.54 0.51

Example 3 In Vivo Anti-Tumor Activity of the Compounds in Animal BodyActivity Against Mouse Grafted Sarcoma S-180

1. The Screened Compounds

Three compounds were selected for the screening test: Compound 6(Curcumol p-hydroxy aniline hydrochloride), Compound 8 (Curcumolpiperazine hydrochloride), and Compound 21 (Curcumol sodium sulfonate).

2. Materials

(1) Experimental animals: Kunming mice weighting 18-22 g;

(2) 100 mg/ml solutions of the 3 compounds were prepared, respectively,in water for injection (1% surface surfactant was added into thesolution of Compound 6). 5-FU and camptothecine, 30 mg/kg and 1 mg/kg,respectively, were used as positive controls;

(3) 0.9% physiological saline;

(4) Tumor cell lines inoculated: sarcoma S-180.

3. Route of Administration: Intravenous Injection.

4. Methods

The experiments were performed according to “Guideline for New Drugs(Western medicines) in Pre-clinical Stages” issued by Bureau of DrugPolicy & Administration of the People's Republic of China, and therelevant methods in “Methodology of Pharmaceutical Experiments”, XuShuyun et al. ed. 120 male Kunming mice weighting 20-22 kg were allowedto grow 3 days. No abnormality was observed. The mice were routinelyinoculated in oxter with mouse sarcoma S-180, and weighted 4 hrs. afterinoculation. The mice were randomly divided into 12 groups, 10mice/group. Group 1 was given 0.3 ml of physiological saline as negativecontrol; Group 2 was given 5-FU as positive control, 0.6 mg/mouse; Group3 was given hydroxycamptothecine as positive control, 0.02 mg/mouse.Groups 4, 5 and 6 were injected with 10% Compound 6, 0.4 ml/mouse, 0.2ml/mouse, and 0.1 ml/mouse, respectively. Groups 7, 8 and 9 wereinjected with 10% Compound 8, 0.4 ml/mouse, 0.2 ml/mouse, and 0.1ml/mouse, respectively. Groups 10, 11, and 12 were injected with 10%Compound 20, 0.4 ml/mouse, 0.2 ml/mouse, and 0.1 ml/mouse, respectively.The tested compounds were administered on the next day afterinoculation, once a day on alternate days, 6 doses totally administered.On the next day after the end dose, the mice were weighed andsacrificed. After the tumor was excised, body weight and tumor weightwere recorded. The tumor inhibition rate was calculated based on thefollowing formula: [(average tumor weight of the control groups−averagetumor weight of the experimental groups)/average tumor weight of thecontrol groups]×100%. The data was processed statistically. The aboveexperiments were repeated three times.

The results are shown in Table 5. The results indicate that all of thethree different concentrations of Compound 6 had a good inhibitoryeffect against mouse sarcoma S-180; Compound 8 had the inhibitory effectonly when the concentration reached 40 mg/kg; while Compound 21 hadlittle inhibitory effect.

TABLE 5 (the first experiment). Results of activity against mousegrafted sarcoma S-180 (X ± SD, n = 10) Dose Average weight(g)- Averageweight (g)- Tumor weight Inhibition Group (mg/kg) before administrationafter administration (g) rate (%) 1 Physiological 20.3 22.1 2.50 ± 0.32 saline 2 5-FU 30 19.5 22.9 0.72 ± 0.23  71.2 3 Hydroxyca-mp 1 20.6 20.80.87 ± 0.28  65.2 tothecine 4 Compound 6 40 19.9 22.0 0.62 ± 0.34  75.25 20 20.3 22.9 0.79 ± 0.21  68.4 6 10 19.6 22.3 1.1 ± 0.31 56 7 Compound8 40 20.3 23.0 1.3 ± 0.24 48 8 20 19.6 21.8 2.0 ± 0.40 20 9 10 19.9 22.52.1 ± 0.42 16 10 Compound 21 40 20.6 23.1 2.3 ± 0.31 8 11 20 19.9 22.52.4 ± 0.43 4 12 10 20.1 22.6 2.6 ± 0.23 —

TABLE 5 (the second experiment). Results of activity against mousegrafted sarcoma S-180 (X ± SD, n = 10) Dose Average weight(g)- Averageweight(g)- Inhibition Group (mg/kg) before administration afteradministration Tumor weight (g) rate (%) 1 Physiological 20.5 22.1 2.60± 0.32  saline 2 5-FU 30 19.2 21.9 0.79 ± 0.33  69.6 3 Hydroxy- 1 20.020.8 0.92 ± 0.38  64.6 camptothecine 4 Compound 6 40 19.5 22.0 0.71 ±0.44  72.7 5 20 20.7 21.9 0.82 ± 0.32  68.8 6 10 20.6 22.3 1.1 ± 0.3157.7 7 Compound 8 40 20.7 23.0 1.4 ± 0.37 46.1 8 20 20.6 21.8 2.2 ± 0.2915.4 9 10 21.0 23.5 2.2 ± 0.31 15.4 10 Compound21 40 20.1 23.1 2.4 ±0.36 7.6 11 20 19.6 22.2 2.5 ± 0.38 3.8 12 10 20.4 22.6 2.5 ± 0.42 3.8

Activity Against Mouse Grafted Liver Cancer H22

The experiments were identical with the experiments for activity againstmouse grafted sarcoma S-180, except that the cell line inoculated wasliver cancer H22. The experiments were repeated three times.

The results are shown in Table 6. The results indicate that all of thethree different concentrations of Compound 6 had a good inhibitoryeffect against mouse grafted liver cancer H22; Compound 8 had someinhibitory effect only when the concentration reached 40 mg/kg; whileCompound 21 had little inhibitory effect.

TABLE 6 (the first experiment). Results of activity against mouse Livercancer cell line H22 (X ± SD, n = 10) Average Average weight(g)-weight(g)- Dose before after Tumor Inhibition Group (mg/kg)administration administration weight (g) rate (%) 1 Physiological 19.622.2 3.02 ± 0.30 saline 2 5-FU 30 20.3 22.3 1.21 ± 0.31 60.0 3 Hydroxy-1 20.3 20.5 1.12 ± 0.28 63.9 camptothecine 4 Compound 6 40 19.9 22.11.15 ± 0.31 61.9 5 20 21.2 22.2 1.60 ± 0.29 47.0 6 10 20.6 22.0  2.8 ±0.37 7.0 7 Compound 8 40 20.8 22.4  1.9 ± 0.36 37.1 8 20 21.0 21.9  2.9± 0.39 4.0 9 10 21.4 22.3 3.02 ± 0.33 — 10 Compound 21 40 20.5 22.1  2.9± 0.31 4.0 11 20 19.2 22.0 3.03 ± 0.41 — 12 10 20.3 22.1 3.00 ± 0.29 —

TABLE 6 (the second experiment). Results of activity against mouse Livercancer cell line H22 (X ± SD, n = 10) Average Average weight(g)-weight(g)- Dose before after Tumor Inhibition Group (mg/kg)administration administration weight (g) rate (%) 1 Physiological 21.222.2 3.02 ± 0.30 saline 2 5-FU 30 19.6 21.3 1.19 ± 0.31 60.5 3 Hydroxy-1 20.3 20.2 1.00 ± 0.28 66.9 camptothecine 4 Compound 6 40 20.8 22.41.21 ± 0.31 60.3 5 20 20.3 22.0 1.58 ± 0.29 47.7 6 10 20.1 21.0  2.6 ±0.37 13.9 7 Compound 8 40 20.4 21.4 1.93 ± 0.36 36.1 8 20 21.8 21.9  2.9± 0.39 4.0 9 10 21.0 22.3 3.10 ± 0.33 — 10 Compound 21 40 20.5 22.1  3.0± 0.31 1.0 11 20 20.3 22.0 3.02 ± 0.41 — 12 10 19.2 22.1 3.03 ± 0.29 —

TABLE 6 (the third experiment). Results of activity against mouse Livercancer cell line H22 (X ± SD, n = 10) Average Average weight (g)- weight(g)- Dose before after Tumor Inhibition Group (mg/kg) administrationadministration weight (g) rate (%) 1 Physiological 20.2 22.0 3.02 ± 0.30saline 2 5-FU 30 19.9 20.3 1.08 ± 0.31 64.2 3 Hydroxy- 1 21.3 22.2 1.05± 0.28 65.3 camptothecine 4 Compound 6 40 19.8 21.4 1.30 ± 0.31 56.9 520 19.3 20.0 1.63 ± 0.29 46.0 6 10 20.5 21.2  2.8 ± 0.37 7.3 7 Compound8 40 21.4 21.9 2.01 ± 0.36 33.4 8 20 19.8 20.9  2.8 ± 0.39 7.3 9 10 19.020.3 2.95 ± 0.33 2.3 10 Compound 21 40 19.5 20.1  3.0 ± 0.31 1.0 11 2019.3 21.0 3.01 ± 0.41 — 12 10 20.2 22.1 3.02 ± 0.29 —

Activity Against Mouse Grafted Uterine Cervix Cancer U14

The experiments were identical with the experiments for activity againstmouse grafted sarcoma S-180, except the following:

(1) the tumor cell line inoculated was mouse grafted uterine cervixcancer U14;

(2) all the mice were female.

The experiments were repeated three times.

The results are shown in Table 7. The results indicate that all thethree concentrations of Compound 6, Compound 8 and Compound 21 had agood inhibitory effect against mouse grafted uterine cervix cancer U14;when the concentration was above 20 mg/kg, the inhibition rates of allthe three compounds against mouse grafted uterine cervix cancer U14 wasabove 50%; when the concentration was above 40 mg/kg, the inhibitionrates of all the three compounds against mouse grafted uterine cervixcancer U14 was above 60%, Compound 6 and Compound 21 even above 70%.

TABLE 7 (the first experiment) Results of the activity against mousegrafted uterine cervix cancer U14 (X ± SD, n = 10) Average Averageweight(g)- weight(g)- Dose before after Tumor Inhibition Groups (mg/kg)administration administration weight (g) rate (%) 1 Physiological 19.222.0 3.51 ± 0.30 saline 2 5-FU 30 20.9 21.3 2.10 ± 0.31 40.2 3 Hydroxy-1 20.3 22.2 1.90 ± 0.28 45.8 camptothecine 4 Compound 6 40 19.1 21.40.96 ± 0.31 72.6 5 20 20.3 21.0 1.23 ± 0.29 65.0 6 10 21.5 22.2  2.2 ±0.37 37.3 7 Compound 8 40 21.0 21.9 1.15 ± 0.36 67.2 8 20 19.2 20.9 1.60± 0.39 54.4 9 10 19.9 20.9 2.26 ± 0.33 35.6 10 Compound 21 40 19.6 21.10.99 ± 0.31 71.8 11 20 19.9 21.0 1.52 ± 0.41 56.7 12 10 20.6 22.1 1.68 ±0.29 52.1

TABLE 7 (the second experiment). Results of the activity against mousegrafted uterine cervix cancer U14 (X ± SD, n = 10) Average Averageweight(g)- weight(g)- Dose before after Tumor Inhibition Groups (mg/kg)administration administration weight (g) rate (%) 1 Physiological 19.822.6 3.40 ± 0.30 saline 2 5-FU 30 20.0 21.3 1.95 ± 0.31 42.6 3 Hydroxy-1 20.6 22.3 1.82 ± 0.28 46.5 camptothecine 4 Compound 6 40 19.9 21.60.90 ± 0.31 73.5 5 20 20.3 21.6 1.20 ± 0.29 64.7 6 10 21.5 22.0  2.3 ±0.37 32.3 7 Compound 8 40 20.3 21.2 1.20 ± 0.36 64.7 8 20 19.6 20.9 1.51± 0.39 55.6 9 10 20.9 21.9 2.08 ± 0.33 38.8 10 Compound 21 40 19.8 21.00.89 ± 0.31 73.8 11 20 19.9 21.1 1.46 ± 0.41 57.1 12 10 20.6 22.5 1.59 ±0.29 53.2

TABLE 7 (the third experiment). Results of the activity against mousegrafted uterine cervix cancer U14 (X ± SD, n = 10) Average Averageweight (g)- weight (g)- Dose before after Tumor Inhibition Groups(mg/kg) administration administration weight (g) rate (%) 1Physiological 19.2 22.2 3.62 ± 0.30 saline 2 5-FU 30 20.5 21.0 2.10 ±0.31 42.0 3 Hydroxy- 1 20.1 21.3 1.96 ± 0.28 45.6 camptothecine 4Compound 6 40 20.1 21.6 1.01 ± 0.31 72.1 5 20 19.3 21.6 1.32 ± 0.29 63.56 10 20.1 22.1 2.32 ± 0.37 35.9 7 Compound 8 40 20.2 21.3 1.25 ± 0.3665.5 8 20 19.9 21.9 1.65 ± 0.39 54.4 9 10 20.1 21.5 2.14 ± 0.33 40.1 10Compound 21 40 19.1 20.1 0.95 ± 0.31 73.7 11 20 19.7 21.0 1.51 ± 0.4158.3 12 10 20.6 22.0 1.64 ± 0.29 54.7

Activity Against Mouse Grafted Lung Cancer Lewis

The experiments were identical with the experiments for activity againstmouse grafted sarcoma S-180, except the following:

(1) the tumor cell line inoculated was mouse lung cancer Lewis;

(2) Compound 6, Compound 8 and Compound 21 were not tested for theiractivity;

(3) the mice were divided into six groups.

The experiments were repeated three times.

The results are shown in Table 8. The results indicate that all thethree different concentrations of Compound 6 had some inhibitory effectagainst mouse lung cancer Lewis, the inhibitory effect being 51.6%,44.8%, 29.4%, respectively. When the concentration was 40 mg/kg, theinhibition rate was comparable with that of camptothecine (positivecontrol), similar to that of 5-FU.

TABLE 8 Results of the activity against mouse lung cancer Lewis (X ± SD,n = 10) Average Average weight (g)- weight (g)- In- Dose before afterTumor hibition Groups (mg/kg) administration administration weight (g)rate (%) (the first experiment). 1 Physiological 20.1 23.2 2.98 ± 0.30saline 2 5-FU 30 19.5 21.0 1.34 ± 0.31 55.0 3 Hydioxy- 1 19.8 21.0 1.50± 0.28 49.7 camptothecine 4 Compound 6 40 20.3 21.6 1.44 ± 0.31 51.7 520 19.6 21.1 1.71 ± 0.29 42.6 6 10 20.2 22.1 2.10 ± 0.37 29.5 (thesecond experiment) 1 Physiological 20.3 22.6 2.41 ± 0.30 saline 2 5-FU30 19.8 21.5 1.10 ± 0.31 54.3 3 Hydroxy- 1 19.1 21.0 1.20 ± 0.28 50.2camptothecine 4 Compound 6 40 19.3 20.6 1.19 ± 0.31 50.6 5 20 19.2 21.81.34 ± 0.29 44.4 6 10 20.9 22.9 1.80 ± 0.37 25.3 (the third experiment)1 Physiological 21.0 24.6 2.70 ± 0.30 saline 2 5-FU 30 21.3 23.5 1.25 ±0.31 53.7 3 Hydroxy- 1 19.9 21.5 1.30 ± 0.28 51.9 camptothecine 4Compound 6 40 21.3 24.6 1.28 ± 0.31 52.6 5 20 21.9 24.8 1.42 ± 0.29 47.46 10 20.6 22.9 1.80 ± 0.37 33.3

Example 4 Screening for In Vitro Activity Against HIV-1 Proteinase(HIV-1 PR)

Principle of the test: HIV-1 proteinase digests the fluorescent-labeledsubstrate in an optimal reaction condition and system. The activity ofthe enzyme can be reflected by the fluorescent intensity in the productof the enzyme reaction. Adding a sample into the reaction system can beused for screening the inhibitor of the enzyme.

Materials and Methods:

1. HIV-1 PR: commercially available, stored at −85° C.

2. Treatment of the samples: Before use, sample was dissolved in DMSO ata suitable concentration, then serially diluted (5×) with doubledistilled water, five serial dilutions per sample.

3. Positive control: indinavir, purchased from GlaxoSmithKline.

4. Methods: The diluted sample was added into the reaction buffercontaining the fluorescent-labeled substrate. Then the geneticengineered target enzyme was added, and incubated in the optimalreaction condition. The fluorescent value was measured with a FLUO starGalaxy luminoscope.

Results:

TABLE 9 Results of primary screening of the activity against HIV-1proteinase. Inhibition rate and IC₅₀ of the compounds at differentconcentrations No. 50 10 2.0 0.40 0.08 IC50 (μg/ml) (μg/ml) (μg/ml)(μg/ml) (μg/ml) (μg/ml) Compound 2 12.66 12.20 2.98 8.64 4.13 — Compound3 5.55 12.06 9.64 13.48 4.98 — Compound 5 1.01 11.22 10.30 9.00 4.38 —Compound 6 7.07 13.15 2.99 1.7 0.89 — Compound 7 1.43 3.51 4.31 6.3214.06 — Compound 8 1.40 0.62 5.27 2.64 9.74 — Compound 17 9.94 6.15−6.00 10.71 3.78 — Compound 20 12.68 9.46 10.57 7.44 8.76 — Compound 215.47 10.16 5.86 14.77 8.74 — Compound 23 1.39 11.23 13.69 11.14 11.88 —Compound 24 −0.63 2.57 11.62 13.48 3.32 — Compound 33 9.14 1.30 10.6311.41 13.21 — Compound 47 0.56 1.78 12.94 12.32 6.89 — 10 nM 92.1 Note:“—”: The initial concentration of the sample inhibits the activity ofHTV-1 proteinase; “*”: The sample itself was fluorescent, whichinterferes the testing system to yield imprecise results.

The results indicate that none of the compounds at a concentration of200 ug/ml had activity against the HTV-1 proteinase.

Example 5 Screening for the In Vitro Activity Against the HIV-1 ReverseTranscriptase

Principle of the test: The template to which the HIV-1 reversetranscriptase interacts was covered onto the enzyme-lined template. Inthe optimal reaction conditions and system, HIV-1 RT will put thesubstrate comprising Biotin-dUTP onto the reaction template. Theactivity of the enzyme was measured as the amount of the integratingBiotin-dUTP in the reaction by using streptavidin-labeled horseradishperoxidase. Adding the sample to the reaction system can identifyinhibitor of the enzyme.

Materials and Methods:

1. HIV-1 RT: commercially available.

2. Treatment of the samples: Before use, the sample was dissolved inDMSO at a suitable concentration, then serially diluted (5×) with doubledistilled water, five serial dilutions per sample.

3. Positive control: Nevirapine (NVP), produced by The Third ChangzhouPharmaceutical Company (Changzhou San Chang).

4. Methods: The diluted sample was added into the reaction buffercontaining Biotin-dUTP and the genetic engineered target enzyme, andincubated in the optimal reaction conditions. A streptavidin-labeledhorseradish peroxidase was added to visual the system, and OD450 wasmeasured.

TABLE 10 Primary screening for the activity against the HIV-1 reversetranscriptase. Initial No. concentration IC50 Compound 2 1/150 of the *stock solution Compound 3 1/150 of the * stock solution Compound 5 200μg/ml — Compound 6 200 μg/ml — Compound 7 200 μg/ml — Compound 8 200μg/ml — Compound 17 200 μg/ml — Compound 20 200 μg/ml — Compound 21 200μg/ml — Compound 23 200 μg/ml — Compound 24 200 μg/ml — Compound 33 200μg/ml — Compound 47 200 μg/ml — NVP  10 μg/ml 0.21 μg/ml Note: “—”: Theinitial concentration of the sample did not inhibit the HIV-1 RT. *: Theinitial concentration of the sample inhibited the HIV-1 RT.

The results shows that Compound 2 and Compound 3 in a concentration of120 ug/ml had 60% inhibition rate of. The new compounds had activityagainst HIV-1 reverse transcriptase, but the effect was barely good.

Example 6 Screening for the Activity Against HIV-1 Integrase (HIV-1 IN)

Principle of the test: A synthetic oligonucleotide of 30 nt was used asthe donor substrate. A synthetic oligonucleotide of 20 nt was used asthe target substrate. A 96-well plate was covered by the donorsubstrate, and a purified HIV-1 integrase was added into each well.ELISA was performed to measure the product of the target DNA chaintransfer. A biotin-labeled alkali phosphatase was used to visual thesystem. OD value was measured by an Enzyme-linked Detector. Add a sampleinto the reaction system for screening the inhibitor of the enzyme.

Materials and Methods:

1. HIV-1 IN: commercially available.

2. Treatment of the samples: Before use, the sample was dissolved inwater or DMSO at a suitable concentration, then serially diluted (5×),four serial dilutions per sample. The stock solutions of two sampleswere 100-fold diluted with DMSO, then 5-fold diluted, four serialdilutions per sample. Positive control: S-y, provided by ShanghaiOrganic Chemistry Institute.

3. The donor substrate and target substrate: synthesized by ShanghaiBiological Engineering Company (Shanghai Shenggong).

4. Methods: The diluted sample was added into a 96-well plate covered bythe donor substrate, and then added into the reaction buffer containingthe genetic engineered target enzyme and biotin-target substrate,incubated in the optimal reaction conditions. A biotin-labeled alkaliphosphatase was used to visual the system to determine the absorbance at450 nm (OD450).

Results:

TABLE 11 Primary screening for activity against the HIV-1 integraseInitial concentration IC₅₀ Initial concentration IC₅₀ No. (μg/ml)(μg/ml) No. (μg/ml) (μg/ml) Compound 2 1/1000 35.681 Compound 100 39.820(Stock solution) 20 Compound 3 1/1000 35.983 Compound 100 — (Stocksolution) 21 Compound 5 100 — Compound 100 — 23 Compound 6 100 —Compound 100 — 24 Compound 7 100 — Compound 100 — 33 Compound 8 100 —Compound 100 — 47 Compound 17 100 — — S-y  0.545 Note: “—”: indicatesthat the initial concentration of the sample had a inhibition rate ofless than 50% against HIV-1 IN.

The results shows that Compound 20 had a good inhibitory effect to HIV-1integrase (HIV-1 IN), IC₅₀ being 39.820 ug/ml.

Example 7 Screening for the Activity Against Influenza A Virus andInfluenza B Virus

Principle of the test: The MDCK (dog kidney) cells are used as host ofvirus to measure the inhibition of the cytopathic effect (CPE) inducedby the virus.

Materials and Methods:

1. Virus strains: influenza A virus (Jifang 90-15) and influenza B virus(Jifang 97-13) were subcultured in the allantoic cavity of a chickembryo (2003.8), stored at −80° C.

2. Treatment of the samples: The sample was dissolved in DMSO, and thendissolved in medium to reach a suitable initial concentration. Theresulting solution was then 3× serially diluted, 8 serial dilutionlevels per sample.

3. Positive: Virazole (RBV), obtained from Zhejing KangyuPharmaceuticals, Co. LTD (Batch No. 960501).

4. Methods: The MDCK cells were seeded into a 96-well plate, andincubated in an atmosphere of 5% CO₂ at 37° C. for 24 hours. 10⁻³(60×TCID₅₀) of influenza A virus and 10⁻² (30×TCID₅₀) of Influenza Bvirus was added to the MDCK cells, respectively. The medium of the viruswas decanted after 2 hrs of incubation at 37° C., and the agents indifferent dilution levels were added. The control for virus and controlfor cells were used. After incubation at 37° C. for 36 hrs, CPE wererecorded, and the 50% inhibiting concentration(IC₅₀) of the samples werecalculated.

Results:

TABLE 12 Primary screening for the activity against influenza A virusand influenza B virus influenza A virus influenza B virus No. TC₅₀(μg/ml) IC₅₀ (μg/ml) SI IC₅₀ (μg/ml) SI 25  >1000 — — — — 21  >100047.72 20.96 — — E >1000 — — — — 6 192.45 — — — — 14  143.17 — — 8 192.45— — — — 2 1/12150 of the stock solution — — — — 3 1/8403 of the stocksolution — — — — RBV >2000 2.06 970.87 6.17 324.15 Note: (1) “—”indicates that the max nontoxic dose of a sample did not have theactivity against influenza A virus or influenza B virus. (2) TC₅₀: theconcentration of 50% toxicity of an agent; IC₅₀: the concentration of anagent with 50% inhibition of a virus; SI: selection index, SI =TC₅₀/IC₅₀.

The results shows that Compound 20 had the activity but the activity isnot potent.

Example 8 Screening for the Agents Against Herpes Virus

Materials and Methods

Experimental samples: The stock solutions were prepared by dissolvingCompounds 3-8, 14, 17, 19, 20, 21, 43 and Curcumol E in PBS or absolutealcohol, respectively, sterilized with a 0.45 μm filter, and stored awayfrom light at 4° C. until use.

Virus and cells: oral membrane herpes virus (VSV), and 293, BHK celllines were stored in liquid nitrogen by the Hepatitis & Gene Therapyresearch group of Wuhan Virus Institute of Chinese Academy of Sciences.Cells were grown in the DMEM medium supplemented with 10% fetal bovineserum (Gibico) in a CO₂ incubator at 37° C.

Preparation of Virus and Test of the Titer: Bhk Cells were Infected withVSV to propagate the virus. After 2-3 days, the cells were centrifuged,and the supernatant was collected. The titer of the virus was measuredwith the TCID₅₀ method, and the virus were stored away from light at−80° C. until use.

Screening for the agents: The 293 cells were counted and 1.5×10⁴cells/well were seeded into a 96-well plate. After incubation overnightin an atmosphere of CO₂ at 37° C., each of the experimental samples in agiven concentration was added to the cells. The cells were infected withVSV of MOI=5×10⁻³. A negative control and a positive control were alsoincluded. The cytopathy were observed 12 hrs and 24 hrs after infection,and photographed with a digital photographer.

Results: Cytopathy and cell status 24 hrs after infection are shown inTable 13.

TABLE 13 Primary screening for the sample against herpes virusconcentration Cytopathy and cell status No. (μg/ml) Sample + VSV SampleCompound 3 200 Cytopathy Normal Compound 4 200 Cytopathy Good Compound 5200 Cytopathy Good Compound 6 200 Cytopathy Good Compound 7 200Cytopathy Good Compound 8 200 Cytopathy Good Compound 14 200 Cytopathy,poor Poor Compound 17 200 Cytopathy Good Compound 19 200 CytopathyApoptotic Compound 20 200 Cytopathy Good Compound 21 200 No remarkableNormal cytopathy Compound 43 200 Cytopathy, Comparatively Comparativelypoor poor E 200 Cytopathy, Comparatively Comparatively poor poor VSV MOI= 0.005 Cytopathy Cytopathy PBS + medium 1% Cytopathy Good alcohol +medium 1% Cytopathy Good

Cytopathy and cell status 12 hrs after partial infection: Since theconcentration used was high (200 μg/ml), which leaded into a hightoxicity to the cells, we decreased the concentration of the sample (50μg/ml) in the following screening experiments. The results are shown inthe following Table 14:

Concentration Cytopathy and cell status No. (μg/ml) Sample + VSV Sample14 50 Cytopathy Good 43 50 Cytopathy Good E 50 Cytopathy Good VSV MOI =0.005 Cytopathy Cytopathy PBS + medium 1% Cytopathy Good Alcohol +medium 1% Cytopathy Good

Cytopathy and status of the cells treated with the primarily screenedsamples 12 hrs and 24 hrs after infection

The results of the first round of the primary screening indicates thatCompound 21 (among the 13 samples) had some activities against VSV.Further screening experiments were performed on Compound 21 at differentconcentrations. The results are as follows:

Cytopathy and cell status Concentration Sample + VSV cell status of No.(μg/ml) 12 h 24 h 12 and 24 h Compound 12.5 Cytopathy Cytopathy Good 2125 Cytopathy Cytopathy Good 50 Cytopathy of Cytopathy Good most cells100 Cytopathy of Cytopathy Good some cells 200 No Cytopathy of Normalsignificant some cells cytopathy

The results shows that Compound 21 in a concentration of 200 μg/ml hadthe activity against VSV.

Example 9 Screening for the Activity Against Hepatitis B Virus (HBV)

1. Materials and Methods

1.1 Experimental samples: The stock solutions were prepared bydissolving Compounds 6, 16, 14, 2, 3, 24, 17, 20, 33, 25, 47, 21 andCurcumol in PBS or absolute alcohol, respectively, sterilized with a0.45 um filter, and stored away from light at 4° C. until use.

1.2 Virus and cells: HBV recombinant Baculovirus (vAcGFPHBVc), sf9 andHepG2 were stored in liquid nitrogen by the hepatitis and gene therapyresearch group of Wuhan Virus Institute of Chinese Academy of Sciences.Cells were grown in the DMEM medium supplemented with 10% fetal bovineserum (Gibico) in a CO₂ incubator (FORMA) at 37° C.

1.3 Preparation of virus and test of the titer: The sf9 cells wereinfected with vAcGFPHBVc to propagate the virus. After 3 days, the cellswere centrifuged, and the supernatant was collected. The titer of thevAcGFPHBVc virus was measured with the TCID₅₀ method, and the virus werestored away from light at −80° C. until use.

1.4 Measurement of HBV e antigen (ELISA method): briefly as follows:

1. The plate and reagents were balanced at 37□ for 30 mins, the samplesto be tested were balanced at room temperature for 30 mins;

2.50 μl of test sample was added into each well. Two well was includedas the negative and positive control, respectively, to which one drop ofthe negative control or positive control was added. There was also onewell as the blank control;

3. One drop of the enzyme-conjugate was added to each well (except theblank well) and then mixed. The plate was covered, and incubated at 37□for 30 mins;

4. The plate was washed by a plate-washing machine, and dried after 5times of washing;

5. One drop of individual Developer A and individual Developer B wererespectively added into each well and mixed. The Plate was covered, andincubated at 37□ for 15 mins;

6. One drop of stopping buffer was added into each well and mixed;

7. The results were read on a Enzyme-linked Detector at 450 nm, andnormalized for results of the blank well.

HBV e antigen diagnostic kit was commercially obtained from ShanghaiShiye Kehua Biotechnology Co. LTD.

1.5 Screening for the agents: The HepG2 cells were counted and 1.5×10⁴cells/well were seeded into a 96-well plate. After incubation overnightin an atmosphere of CO₂ at 37° C., each of the experimental samples in agiven concentration was added to the cells. After incubation at 37□ for24 hrs, the HepG2 treated with the samples were infected with vAcGFPHBVcof 100 MOI. A negative control and a positive control were alsoincluded. Four days after infection, the growth of the cells wereobserved, and the HBV e antigen in the supernatant was measured with theHBV e antigen diagnostic kit.

2. Results

2.1 Results of the HBV e antigen

The primary screening excluded the influence of the agents to the cells.The results primarily indicates that Compound 6, 16 and 2 amongst 13samples had the activity against HBV.

(Value of the positive control - OD₄₅₀ Measured Value) Concentration ofValue of the positive No. (μg/ml) Cell status HBeAg control × 100%  6200 Normal 0.126 93.42723 16 200 Normal 0.502 73.81325 14 200 Poor 0.05397.23516  2 200 Normal 0.607 68.33594  3 200 Normal 2.61 −36.1502 24 200Normal 2.387 −24.5179 17 200 Poor 0.073 96.19197 20 200 Normal 2.201−14.8148 33 200 Poor 0.068 96.45279 Curcumol 200 Normal 2.654 −38.445525 200 Poor 2.527 −31.8206 47 200 Poor 0.365 80.95983 21 200 Retarded0.137 92.85342 growth + Normal 1.917 − Normal 0.057

FIG. 1 shows the measured OD₄₅₀ value of the HBV e antigen sample,wherein the X-axis represents the No. of the test sample, whichcorresponds to the compounds of the present invention as follows:

No. in FIG. 1: 1 2 3 4 5 6 7 A B C D E F No. of the compounds 6 16 14 23 24 17 20 33 Curcumol 25 47 21

POSITIVE means the positive control, and NEGATIVE means the negativecontrol.

FIG. 2 shows the decreased HbeAg by percent of the samples relative tothat of the positive control. The numbering of FIG. 2 is identical withthat of FIG. 1.

1. A compound or a pharmaceutically acceptable salt thereof, wherein said compound comprising: a curcumol molecule modified by chemical groups Y or R¹ as shown in formula I:

wherein, the chemical group R¹ is selected from the group consisting of R, RCO, HO₃S, acyl of coffeic acid, gambogic acid, isogambogic acid, neogambogic acid and glycyrrhizic acid; Y is selected from the group consisting of Y¹NY², Y¹CONY², ═CHR², —CH₂R²,

 —OH and —OR, wherein Y¹ is H or C₁₋₈, Y² is a C₁₋₈ heterocycle selected from the group consisting of

wherein P is S, O or N, Z is selected from the group consisting of H, hydroxy group, saturated or unsaturated linear C₁₋₆ hydrocarbon group, saturated or unsaturated branched C₃₋₆ hydrocarbon group, n=1-3, R is selected from the group consisting of H, saturated or unsaturated linear C₁₋₁₀ hydrocarbon group, saturated or unsaturated branched C₃₋₁₀ hydrocarbon group, C₃₋₁₀ hydrocarbon ether, C₃₋₁₀ hydrocarbon sulfide, saturated or unsaturated C₃₋₈ cyclic hydrocarbon group optically substituted by at least one substituent selected from the group consisting of nitro, sulfonic acid group, halogen atom, hydroxyl group and C₆₋₁₂ aryl group, R² is selected from the group consisting of F, Cl, Br, I, —OH, —OR, —HSO₃, —NO₃, RNH—, R′NR″, pyridyl, pyrrolyl, imidazolyl, triazolyl, tetrazolyl, dioxazolyl, dioxadiazolyl, piperidyl,

wherein, R′ is selected from the group consisting of H, saturated or unsaturated linear C₁₋₁₀ hydrocarbon group, saturated or unsaturated branched C₃₋₁₀ hydrocarbon group, C₃₋₁₀ hydrocarbon ether, C₃₋₁₀ hydrocarbon sulfide, saturated or unsaturated C₃₋₈ cyclic hydrocarbon group optically substituted by at least one substituent selected from the group consisting of nitro, sulfonic acid group, halogen atom, hydroxyl group, C₆₋₁₂ aryl group and H₂NRNH, R″ is selected from the group consisting of H, saturated or unsaturated linear C₁₋₁₀ hydrocarbon group, saturated or unsaturated branched C₃₋₁₀ hydrocarbon group, C₃₋₁₀ hydrocarbon ether, C₃₋₁₀ hydrocarbon sulfide, saturated or unsaturated C₃₋₈ cyclic hydrocarbon group optically substituted by at least one substituent selected from the group consisting of nitro, sulfonic acid group, halogen atom, hydroxyl group, C₆₋₁₂ aryl group and H₂NRNH.
 2. The compound of claim 1, wherein the aryl group is selected from the group consisting of Ar—, ArCH₂—, ArCH₂CH₂—, and CH₃ArCH₂CH₂—, wherein Ar— is phenyl or phenyl group substituted with F, Cl, Br, I, nitro, sulfonic group or 1-3 hydroxy groups.
 3. A compound or a pharmaceutically acceptable salt thereof, wherein said compound comprising formula III:

wherein R² is H, and R¹ is HO₃S, propionyl, butyryl, isobutyryl or benzoyl.
 4. A compound or a pharmaceutically acceptable salt thereof, wherein said compound comprising formula III:

wherein R¹ and R² are as shown the following table: Name of the compounds R¹ R² Curcumol butyrate CH₃CH₂CH₂CH₂C— H Curcumol isobutyrate (CH₃)₂CH₂CO— H Curcumol benzoate

H Curcumol p-hydroxy aniline H

Curcumol p-hydroxy aniline hydrochloride H

Curcumol piperazine H

Curcumol piperazine hydrochloride H

Curcumol heterocyclyl ethylamine H

Curcumol heterocyclyl ethylamine hydrochloride H

3,4-dihydroxy aniline H

3,4-dihydroxy aniline hydrochloride H

Curcumol n-butyl amine H CH₃CH₂CH₂CH₂NH— Curcumol n-butyl amine H CH₃CH₂CH₂CH₂NH— hydrochloride Curcumol t-butyl amine H (CH₃)₃CNH— Curcumol t-butyl amine H (CH₃)₃CNH— hydrochloride Curcumol monobromide H —Br Curcumol monohydroxy H —OH compound Curcumol mononitrate H —NO₃ Curcumol sulfonate HSO₃— H Curcumol sodium NaSO₃— H sulfonate Curcumol acrylate CH₂═CHCO— H Curcumol diethanolamine H (CH₃CH₂)₂N— Curcumol diethanolamine H (CH₃CH₂)₂N— hydrochloride Curcumol methyl ether CH₃ —Br bromide Curcumol methyl ether CH₃ CH₃CH₂CH₂CH₂NH— n-butyl amine Curcumol methyl ether CH₃ CH₃CH₂CH₂CH₂NH— n-butyl amine hydrochloride Curcumol ethyl ether CH₃CH₂— —NO₃ nitrate Curcumol propionate CH₃CH₂CO— H.


5. The compound of claim 1, wherein, Y . . . is a single bond, Y is —OH or —OR¹.
 6. The compound of claim 1, wherein R¹ is H, Y is selected from Y¹NY², Y¹CONY², ═CHR², —CH₂R²,

—OH.
 7. A compound or a pharmaceutically acceptable salt thereof, wherein said compound comprising Formula II:

wherein R¹, R² and R³ are as shown the following table: Name of the compounds R¹ R² R³ Curcumol dibromide H —Br —Br Curcumol dinitrate H —NO₃ —NO₃ Curcumol dihydroxy compound H —OH —OH Curcumol monobromide without H H —Br double bond Curcumol monohydroxy compound H H —OH without double bond Curcumol mononitrate without H H —NO₃ double bond Curcumol p-hydroxy aniline without double bond H H

Curcumol p-hydroxy aniline hydrochloride without double bond H H

Curcumol bis (p-hydroxy aniline) H

Curcumol bis (p-hydroxy aniline) hydrochloride H

Curcumol bispiperazine H

Curcumol bispiperazine hydrochloride H

Curcumol piperazine without double bond H H

Curcumol piperazine hydrochloride without double bond H H

Curcumol methyl ether n-butyl CH₃ H CH₃CH₂CH₂CH₂NH amine without double bond Curcumol methyl ether n-butyl CH₃ H CH₃CH₂CH₂CH₂NH amine hydrochloride without double bond.


8. An anti-tumor pharmaceutical composition comprising a pharmaceutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claim 1, 2, 3, 4, 5, 6 or 7, and a pharmaceutically acceptable excipient or an additive, wherein said compound inhibits tumor cell growth in vitro, wherein the tumor cell is selected from the group consisting of human histocytic lymphoma, human lung adenocarcinoma, human hepatic carcinoma, human mammary adenocarcinoma, human cervical carcinoma, human promyelocytic leukemia, and mouse Lewis lung carcinoma.
 9. An anti-cancer pharmaceutical composition comprising a pharmaceutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claim 1, 2, 3, 4, 5, 6 or 7, and a pharmaceutically acceptable excipient or an additive, wherein the cancer is selected from the group consisting of sarcoma, liver cancer, lung cancer and uterine cervix cancer.
 10. An anti-virus pharmaceutical composition comprising a pharmaceutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claim 1, 2, 3, 4, 5, 6 or 7, and a pharmaceutically acceptable excipient or an additive, wherein said compound inhibits viral enzyme activity or virus replication, said virus is selected from the group consisting of HIV, influenza A virus, infuluenza B virus, herpes virus and hepatitis B virus.
 11. A method of inhibiting tumor cell growth comprising administering a pharmaceutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claim 1, 2, 3, 4, 5, 6 or 7 to a mammal to inhibit the growth of tumor cell in the mammal.
 12. The method of claim 11, wherein said tumor is selected from the group consisting of sarcoma, liver cancer, lung cancer and uterine cervix cancer.
 13. A method of inhibiting tumor cell growth comprising administering a pharmaceutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claim 1, 2, 3, 4, 5, 6 or 7 to a mammal, wherein the tumor cell is selected from the group consisting of human histocytic lymphoma, human lung adenocarcinoma, human hepatic carcinoma, human mammary adenocarcinoma, human cervical carcinoma, human promyelocytic leukemia, and mouse Lewis lung carcinoma.
 14. A method of inhibiting virus growth comprising administering a pharmaceutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claim 1, 2, 3, 4, 5, 6 or 7 to a mammal to inhibit viral enzyme activity or virus replication of said virus, wherein said virus is selected from the group consisting of HIV, influenza A virus, infuluenza B virus, herpes virus or hepatitis B virus. 